Non-human animals expressing humanized cd3 complex

ABSTRACT

Non-human animals, expressing humanized CD3 proteins are provided. Non-human animals, e.g., rodents, genetically modified to comprise in their genome humanized CD3 proteins are also provided. Additionally, provided are methods and compositions of making such non-human animals, as well as methods of using said non-human animals.

FIELD OF INVENTION

A genetically modified non-human animal (e.g., a rodent, e.g., a mouseor a rat) is provided that comprises in its genome a nucleic acidsequence encoding a humanized CD3 protein, e.g., a humanized CD3ε, ahumanized CD3δ, and/or humanized CD3γ. Thus, genetically modifiednon-human animals that express humanized CD3 complex are provided. Alsoprovided herein is a model for preclinical testing of CD3-basedtherapeutics, e.g., CD3-based antibodies, e.g., CD3-based bispecificantibodies.

BACKGROUND OF THE INVENTION

In addition to the T cell receptor subunits, e.g., highly variable TCRαand TCRβ, the T cell receptor complex on the surface of a T cellcomprises invariant CD3ε, CD3δ, and CD3γ chains, which form heterodimersconsisting of CD3εδ and CD3εγ. Also associated with the TCR/CD3 complexis the ζ chain, which is present as a disulfide-linked homodimer.

CD3 chains play a crucial role in T cell receptor assembly, transport tothe cell surface, endocytosis of surface receptors, T cell development,and T cell signaling. For example, it has been demonstrated throughstudies of deficiencies of various CD3 subunits, that CD3 chains areimportant for double negative (CD4−CD8− or DN) to double positive(CD4+CD8+ or DP) to single positive (CD4+ or CD8+ or SP) T celltransition. In addition, each of CD3ε, CD3δ, and CD3γ chains containsone immunoreceptor tyrosine-based activation motif (ITAM) while the ζchain dimer contains 6 total ITAMs. These motifs serve as signalingmodules, and are phosphorylated by associated kinases upon TCRengagement.

Antibodies against CD3 have been shown to cluster CD3 on T cells,thereby causing T cell activation in a manner similar to the engagementof the TCR by peptide-loaded MHC molecules. Thus, anti-CD3 antibodieshave been proposed as therapeutic candidates aimed at activation of Tcells. In addition, bispecific antibodies that are capable of bindingCD3 and a target antigen have been proposed for therapeutic usesinvolving targeting T cell immune responses to tissues and cellsexpressing the target antigen.

A convenient animal model for preclinical testing of mono- andbispecific CD3-based therapeutic antibodies is particularly desired.

SUMMARY OF THE INVENTION

Provided herein is a genetically modified non-human animal comprising atan endogenous non-human CD3 locus a nucleic acid sequence encoding anextracellular domain of human CD3 protein, wherein the human CD3 proteinis selected from the group consisting of CD3ε, CD3δ, CD3γ, CD3ζ, and acombination thereof. In one embodiment, the non-human animal comprisesat the endogenous non-human CD3 locus a nucleic acid sequence encodingan extracellular domain of human CD3ε, an extracellular domain of humanCD3δ, and an extracellular domain of human CD3γ. In one embodiment, theanimal does not comprise a functional extracellular domain(s) of thecorresponding non-human protein(s). In one embodiment, the animalfurther comprises a nucleic acid sequence(s) encoding transmembrane andcytoplasmic domains of corresponding endogenous non-human animal CD3protein(s). In one embodiment, the nucleic acid sequence(s) encoding theextracellular domain of the human CD3 in the non-human animal isoperably linked to the nucleic acid sequence(s) encoding transmembraneand cytoplasmic domains of the corresponding endogenous non-human animalCD3 protein(s). In a particular embodiment, the non-human animalcomprises (a) at an endogenous CD3ε locus a nucleic acid sequenceencoding an extracellular domain of a human CD3ε operably linked to anucleic acid sequence encoding transmembrane and cytoplasmic domains ofan endogenous non-human animal CD3ε, (b) at an endogenous CD3δ locus anucleic acid sequence encoding an extracellular domain of a human CD3δoperably linked to a nucleic acid sequence encoding transmembrane andcytoplasmic domains of an endogenous non-human animal CD3δ, and (c) atan endogenous CD3γ locus a nucleic acid sequence encoding anextracellular domain of a human CD3γ operably linked to a nucleic acidsequence encoding transmembrane and cytoplasmic domains of an endogenousnon-human animal CD3γ. In some embodiments, the extracellular domain ofthe human CD3 protein in the non-human animal comprises the sequenceselected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, andSEQ ID NO:35. In some embodiments, the animal comprises extracellulardomains of human CD3 proteins which comprise the sequences of SEQ IDNO:33, SEQ ID NO:34 and SEQ ID NO:35.

In a particular embodiment, the non-human animal provided is a mammal.In one embodiment, the animal is a rodent. In one embodiment, the animalis a rat or a mouse. In one embodiment, the animal is a mouse. Thus, inone embodiment, provided herein is a genetically modified mouse, whereinthe mouse comprises (a) at an endogenous mouse CD3ε locus a nucleic acidsequence encoding an extracellular domain of a human CD3ε operablylinked to a nucleic acid sequence encoding transmembrane and cytoplasmicdomains of an endogenous mouse CD3ε, (b) at an endogenous mouse CD3δlocus a nucleic acid sequence encoding an extracellular domain of ahuman CD3δ operably linked to a nucleic acid sequence encodingtransmembrane and cytoplasmic domains of an endogenous mouse CD3δ, and(c) at an endogenous mouse CD3γ locus a nucleic acid sequence encodingan extracellular domain of a human CD3γ operably linked to a nucleicacid sequence encoding transmembrane and cytoplasmic domains of anendogenous mouse CD3γ, and the mouse expresses humanized CD3ε, CD3δ, andCD3γ proteins on the surface of its T cells. In one embodiment, theamino acid sequence of the humanized CD3ε protein in said mouse is setforth in SEQ ID NO:24, the amino acid sequence of the humanized CD3δprotein is set forth in SEQ ID NO:25, and the amino acid sequence of thehumanized CD3γ protein is set forth in SEQ ID NO:26. In one embodiment,the mouse displays similar CD4+ to CD8+ cell ratio in the thymus ascompared to a mouse that is not genetically modified to expresshumanized CD3 proteins. In one embodiment, the mouse CD4+ to CD8+ T cellratio in the thymus that is within 30%, within 25%, within 20%, within15%, within 12%, within 10%, within 5%, or within 2% of the CD4+ to CD8+cell ratio of a mouse that is not genetically modified to expresshumanized CD3 proteins. In one embodiment, the mouse displays similar Tand B cell percentages in spleen, lymph nodes, and peripheral blood as amouse that is not genetically modified to express humanized CD3proteins. In one embodiment, the mouse displays similar numbers ofcirculating white blood cells, lymphocytes, monocytes, neutrophils,eosinophils, and basophils as a mouse that is not genetically modifiedto express humanized CD3 proteins.

Thus, in one aspect provided herein is a genetically modified mousecomprising at an endogenous mouse CD3 locus a nucleic acid sequenceencoding an extracellular domain of human CD3 protein, wherein the humanCD3 protein is selected from the group consisting of CD3ε, CD3δ, CD3γ,CD3ζ, and a combination thereof. In one embodiment, the mouse comprisesextracellular domains of human CD3ε, CD3δ, and CD3γ. In one embodimentof the mouse, the extracellular domain of human CD3ε is set forth in SEQID NO:33, the extracellular domain of human CD3δ is set forth in SEQ IDNO:34, and the extracellular domain of human CD3γ is set forth in SEQ IDNO:35. In one embodiment, the mouse expresses a humanized CD3ε, ahumanized CD3δ, and a humanized CD3γ. In one embodiment of the mouse,the humanized CD3ε is set forth in SEQ ID NO:24, the humanized CD3δ isset forth in SEQ ID NO:25, and the humanized CD3γ is set forth in SEQ IDNO:26. In one embodiment, the mouse further comprises mouse CD3ε, CD3δ,and CD3γ transmembrane and cytoplasmic domains. In one embodiment, themouse further comprises endogenous mouse CD3ε, CD3δ, and CD3γtransmembrane and cytoplasmic domains.

In another aspect, provided herein is a method of making a geneticallymodified non-human animal expressing a humanized CD3 protein, comprisingplacing at an endogenous non-human animal CD3 locus a nucleic acidsequence encoding an extracellular domain of human CD3 protein, whereinthe human CD3 protein is selected from the group consisting of CD3ε,CD3δ, CD3γ, CD3ζ, and a combination thereof. In one embodiment of themethod, the animal does not comprise a functional extracellulardomain(s) of the corresponding non-human protein(s). In one embodimentof the method, the animal comprises at the endogenous CD3 locus anucleic acid sequence encoding an extracellular domain of human CD3ε, anextracellular domain of human CD3δ, and an extracellular domain of humanCD3γ. In one embodiment of the method, the extracellular domain of humanCD3ε is set forth in SEQ ID NO:33, the extracellular domain of humanCD3δ is set forth in SEQ ID NO:34, and the extracellular domain of humanCD3γ is set forth in SEQ ID NO:35. In one embodiment of the method, theanimal does not comprise functional extracellular domain(s) of thecorresponding non-human protein(s). In one particular embodiment, themethod comprises replacing at the endogenous CD3 locus an extracellulardomain of a non-human CD3 protein(s) with a corresponding extracellulardomain of a human CD3 protein(s). In one embodiment of the method, theanimal further comprises a nucleic acid sequence(s) encodingtransmembrane and cytoplasmic domains of corresponding endogenousnon-human animal CD3 protein(s). In one embodiment of the method, thenon-human animal is a mouse and a replacement is at the endogenous mouseCD3 locus. In one embodiment of the method wherein the animal is amouse, the mouse expresses a humanized CD3 protein selected from thegroup consisting of a humanized CD3ε set forth in SEQ ID NO: 24, ahumanized CD3δ set forth in SEQ ID NO:25, a humanized CD3γ set forth inSEQ ID NO:26, and a combination thereof. In one embodiment of themethod, the replacement is made in a single ES cell, and the single EScell is introduced into the mouse embryo to make a mouse.

In yet another aspect, provided herein is a mouse model for testing aCD3-based bispecific antigen-binding protein, wherein theantigen-binding protein is capable of binding both CD3 and an antigen ofinterest, the mouse model comprising (a) a nucleic acid sequenceencoding a humanized CD3 protein, wherein the humanized CD3 protein isselected from the group consisting of CD3ε, CD3δ, CD3γ, CD3ζ, and acombination thereof, and wherein the mouse comprises a T cell expressingsaid humanized CD3 protein, and (b) a cell expressing or comprising theantigen of interest. In one embodiment of the mouse model, the nucleicacid sequence(s) of the humanized CD3 protein(s) is located at theendogenous CD3 locus. In one embodiment of the mouse model, theantigen-binding protein has been introduced into said mouse. In oneembodiment of the mouse model, the mouse expresses human CD3ε, CD3δ, andCD3γ extracellular domains. In one embodiment of the mouse model, themouse further expresses mouse CD3ε, CD3δ, and CD3γ transmembrane andcytoplasmic domains.

In one embodiment of the mouse model, the mouse comprises a xenograft ofa tumor expressing the antigen of interest. In one embodiment of themouse model, the cell expressing or comprising the antigen of interestis a tumor cell. In one embodiment of the mouse model, the bispecificantigen-binding protein selected binds to both the humanized CD3 proteinand the antigen of interest. In one embodiment of the mouse model, theantigen of interest is a human antigen. In one embodiment of the mousemodel, the antigen binding protein is capable of binding a monkey CD3protein. In one embodiment of the mouse model, the antigen of interestis a tumor associated antigen. In such an embodiment, the tumorassociated antigen may be selected from the group consisting of AFP,ALK, BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonicanhydrase IX, caspase-8, CCR5, CD19, CD20, CD30, CD40, CDK4, CEA, CTLA4,cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML,EpCAM, EphA2, Fra-1, FOLR1, GAGE protein, GD2, GD3, GloboH, glypican-3,GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGEprotein, MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16(CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53,PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGEproteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, survivin,TAG-72, TGF-β, TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase, and uroplakin-3.

In another embodiment, the antigen of interest is an infectious diseaseassociated antigen. In such an embodiment, the mouse may be infectedwith an infectious agent. In one such embodiment, the infectious diseaseassociated antigen may be a viral antigen and the viral antigen isselected from the group consisting of HIV, hepatitis A, hepatitis B,hepatitis C, herpes virus (e.g., HSV-1, HSV-2, CMV, HAV-6, VZV, EpsteinBarr virus), adenovirus, influenza virus, flavivirus, echovirus,rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus,mumps virus, rotavirus, measles virus, rubella virus, parvovirus,vaccinia virus, HTLV, dengue virus, papillomavirus, molluscum virus,poliovirus, rabies virus, JC virus, ebola virus, and arboviralencephalitis virus antigen. In another such embodiment, the infectiousdisease associated antigen may be a bacterial antigen and the bacterialantigen is selected from the group consisting of chlamydia, rickettsia,mycobacteria, staphylococci, streptococci, pneumonococci, meningococci,gonococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospira, and Lyme disease bacterial antigen.

In one embodiment of the provided mouse model, the CD3-basedantigen-binding protein is an antibody. In one embodiment, the CD3-basedantigen-binding protein is a human or humanized antigen-binding protein.Such mouse model may allow testing for efficacy and/or toxicity of theantigen-binding protein in the mouse.

Also provided herein is a method of screening drug candidates thattarget an antigen of interest comprising (a) providing or receiving agenetically modified mouse comprising at its endogenous mouse CD3 locusa nucleic acid sequence encoding an extracellular domain of a human CD3protein selected from the group consisting of CD3ε, CD3δ, CD3γ, CD3ζ,and a combination thereof, wherein the mouse expresses a functionalhumanized CD3 protein on the surface of its T cells, (b) introducinginto the said genetically modified mouse the antigen of interest, (c)contacting said mouse with a drug candidate of interest, wherein thedrug candidate is directed against the human CD3 and the antigen ofinterest, and (d) determining if the drug candidate is efficacious inpreventing, reducing or eliminating cells characterized by the presenceor expression of the antigen of interest. In one embodiment of themethod, the genetically modified mouse comprises at the endogenous mouseCD3 locus a nucleic acid sequence encoding an extracellular domain ofhuman CD3ε, an extracellular domain of human CD3δ, and an extracellulardomain of human CD3γ. In one embodiment of the method, the mouse doesnot comprise a functional extracellular domain of the correspondingmouse protein(s). In one embodiment of the method, the mouse comprises anucleic acid sequence(s) encoding transmembrane and cytoplasmic domainsof corresponding endogenous mouse CD3 protein(s). In one embodiment ofthe method, the nucleic acid sequence(s) encoding the extracellulardomain of the human CD3 is operably linked to the nucleic acidsequence(s) encoding transmembrane and cytoplasmic domains of thecorresponding endogenous mouse CD3 protein(s). In one embodiment of themethod, the extracellular domain of a human CD3ε is set forth in SEQ IDNO:33, the extracellular domain of a human CD3δ is set forth in SEQ IDNO:34, and the extracellular domain of a human CD3γ is set forth in SEQID NO:35. Thus, in one particular embodiment of the method, the mouseexpresses a humanized CD3ε protein comprising an amino acid sequence setforth in SEQ ID NO:24, a humanized CD3δ protein comprising an amino acidsequence set forth in SEQ ID NO:25, and a humanized CD3γ proteincomprising an amino acid sequence set forth in SEQ ID NO:26.

In a particular embodiment of the method of screening drug candidatesdescribed herein, the step of introducing the antigen of interest intothe mouse described herein comprises expressing in the mouse the antigenof interest. In one embodiment, the step of expressing in the mouse theantigen of interest comprises genetically modifying the mouse to expressthe antigen of interest. In one embodiment, the step of introducing theantigen of interest comprises infecting the mouse with the antigen ofinterest. In one embodiment of the method, the step of introducingcomprises introducing into said mouse a cell expressing the antigen ofinterest. In various embodiments of the method, the cell can be a tumorcell, a bacterial cell, or a cell infected with a virus. Thus, in someembodiments of the method, the mouse comprises and infection which iseither a viral or bacterial infection. Thus, the antigen of interest canbe an infectious disease associated antigen. In one embodiment, theantigen of interest is a viral antigen, and the viral antigen isselected from the group consisting of HIV, hepatitis A, hepatitis B,hepatitis C, herpes virus (e.g., HSV-1, HSV-2, CMV, HAV-6, VZV, EpsteinBarr virus), adenovirus, influenza virus, flavivirus, echovirus,rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus,mumps virus, rotavirus, measles virus, rubella virus, parvovirus,vaccinia virus, HTLV, dengue virus, papillomavirus, molluscum virus,poliovirus, rabies virus, JC virus, ebola virus, and arboviralencephalitis virus antigen. In another embodiment, the antigen ofinterest is an infectious disease associated antigen, which is abacterial antigen selected from the group consisting of chlamydia,rickettsia, mycobacteria, staphylococci, streptococci, pneumonococci,meningococci, gonococci, klebsiella, proteus, serratia, pseudomonas,legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism,anthrax, plague, leptospira, and Lyme disease bacterial antigen.

In another embodiment of the method of screening drug candidates, theantigen of interest is a tumor associated antigen. In one embodiment ofthe method, the tumor associated antigen is selected from the groupconsisting of AFP, ALK, BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS,CA9, carbonic anhydrase IX, caspase-8, CCR5, CD19, CD20, CD30, CD40,CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3,ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein, GD2, GD3,GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3,hTERT, LMP2, MAGE protein, MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3,Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85,NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME,PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1,Steap-2, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase,and uroplakin-3.

In some embodiments of the method of screening drug candidates, themouse is an immunocompetent mouse. In some embodiments of the methoddescribed herein, the antigen of interest is a human antigen ofinterest.

In some embodiments of the method, the drug candidate is an antibody. Insome embodiments, the drug candidate is an antigen-binding protein. Insome embodiments, wherein the drug candidate is an antibody, theantibody is a bispecific antibody or a bispecific antigen bindingprotein. In some embodiments, the bispecific antigen binding protein iscapable of binding both human CD3 protein and the antigen of interest.In one embodiment, the drug candidate is capable of recognizing a monkeyCD3 protein.

In some embodiments of the method of screening drug candidates, the drugcandidate is capable of reducing, eliminating, or preventing tumorgrowth as compared to an agent that does not target the antigen ofinterest. In some embodiments of such method the step of determining ifthe drug candidate is efficacious in preventing, reducing or eliminatingcells characterized by the presence or expression of the antigen ofinterest comprises a tumor volume assay or a T cell mediated tumor cellkilling assay.

In other embodiments, the drug candidate is capable of reducing,eliminating, or preventing bacterial or viral infection as compared toan agent that does not target the antigen of interest. In some suchembodiments, the step of determining if the drug candidate isefficacious in preventing, reducing or eliminating cells characterizedby the presence or expression of the antigen of interest comprises themeasurement of viral or bacterial titers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of T cell receptor complex. The complexcomprises two CD3ε subunits, one CD3δ subunit, one CD3γ subunit, and twoCD3ζ subunits, complexed with the TCRαβ heterodimer on a T cell surface.Asterisks indicate the locations of the ITAM motifs.

FIG. 2 is the schematic representation (not to scale) of the humanizedCD3γδε large targeting vector. FIG. 2A depicts the large targetingvector before the selection cassette (Neo) deletion, with human CD3E,CD3D, and CD3G sequence knock-in locations indicated. A, B, C, D, E, F,and G indicate location of the junction nucleic acid sequencesrepresented in Table 1. FIG. 2B depicts the large targeting vector afterdeletion of the selection cassette (Neo); similarly to FIG. 2A,locations of the human CD3E, CD3D, and CD3G are indicated. A-B, C, D, E,F, and G are locations of the junction nucleic acid sequencesrepresented in Tables 1 and 3.

FIG. 3 depicts the amino acid sequences of the humanized CD3 proteins inthe humanized CD3γδε mice. The CD3 protein sequences of human origin areunderlined.

FIG. 4 depicts alignments between mouse and human CD3e, CD3d, and CD3gsequences. The 5′ and 3′ ends of the human sequences that wereintroduced into mouse CD3 loci are marked with * and **, respectively.

FIG. 5, top row, is a FACs analysis plot demonstrating normaldistribution of CD4+ and CD8+ thymocytes in wild type (WT), heterozygoushumanized CD3γδε (HET), or homozygous humanized CD3γδε (H0) mice. Themiddle row is data depicting percentages as well as numbers of B and Tcells in peripheral blood of indicated animals. Bottom row is datadepicting percentages of T and B cells in the spleen of indicatedanimals.

FIG. 6 is a demonstration of viral LCMV titers in the spleens of eitherwild type control or humanized CD3γδε mice in mice infected with LCMVClone 13 (FIG. 6A), or LCMV clone 13 following prior LCMV Armstrongclone infection (FIG. 6B).

FIG. 7 is data from the FACS analysis of splenocytes from wild type(WT), heterozygous humanized CD3γδε (hCD3γδε Het), or homozygoushumanized CD3γδε (hCD3γδεHo) mice sorted with two anti-human CD3antibodies that also cross-react with monkey CD3 (ah/mfCD3-2 andah/mfCD3-1), two anti-human CD3 antibodies that are human CD3 specific(ahCD3-1 and ahCD3-2), control anti-mouse CD3 (amCD3-2C11), unrelatedcontrol human IgG (control hIgG) and secondary only antibody control(2^(nd) only). MFI values are listed in the tables below each graph.

FIGS. 8A-B demonstrate response to anti-CD3 antibodies in humanizedCD3γδε mice. FIG. 8A demonstrates transient T and B cell depletion inblood of mice treated with anti-CD3 antibodies; either T cell depletionon day 1 for each antibody indicated (left figure), or T and B celldepletion and recovery over 14 days for each antibody tested (middle andright figures). FIG. 8B depicts an increase in concentration ofcytokines released (IFNγ, KC, TNFα, IL-6, and IL-10) 2 hours aftertreatment with indicated antibodies.

FIG. 9 demonstrates splenocytes proliferation (measured as foldactivation over cells only) upon treatment with increasing amounts ofindicated antibodies in wild type (WT) and humanized CD3γδε homozygous(hCD3γδε Ho) mice.

FIG. 10 is a table summarizing various properties of the humanized CD3mouse model.

FIG. 11A demonstrates the effect of anti-CD3 antibody (Ab-1; bispecificantibody recognizing CD3 and CD20, tested at two differentconcentrations) on tumor volume of B16F10.9/CD20 tumors when treatmentis initiated at the same time as tumor implantation (prophylacticmodel). FIG. 11B demonstrates the effect of anti-CD3 antibody (Ab-1;bispecific antibody recognizing CD3 and CD20, tested at two differentconcentrations) on tumor volume of already established B16F10.9/CD20tumors (therapeutic model).

DETAILED DESCRIPTION Definitions

The present invention provides genetically modified non-human animals,e.g., rodents, e.g., mice or rats, that express humanized CD3 proteins,e.g., humanized CD3ε, CD3δ, CD3γ, and/or CD3ζ proteins. The presentinvention also relates to genetically modified non-human animals thatcomprise in their genome, e.g., in their germline, genetically modifiedCD3 loci encoding humanized CD3 proteins, e.g., chimeric human/mouse CD3proteins. Also provided are embryos, cells, and tissues comprising thesame, methods of making the same, as well as methods of using the same.Unless defined otherwise, all terms and phrases used herein include themeanings that the terms and phrases have attained in the art, unless thecontrary is clearly indicated or clearly apparent from the context inwhich the term or phrase is used.

“CD3,” as used herein, includes an antigen which is expressed on T cellsas part of the multimolecular T cell receptor (TCR) complex and whichexists as a homodimer or heterodimer formed from the association of twoof four receptor chains: CD3-epsilon (ε), CD3-delta (δ), CD3-zeta (ζ),and CD3-gamma (γ). Sequences and GenBank Accession Numbers of human andmouse CD3-delta, CD3-zeta, and CD3-gamma are presented in Table 4 below.Throughout the application, ε or epsilon can also be written as E, δ ordelta can also be written as D, ζ or zeta can also be written as Z, andγ or gamma can also be written as G.

As used herein, “an antibody that binds CD3” or an “anti-CD3 antibody”includes antibodies and antigen-binding fragments thereof thatspecifically recognize a single CD3 subunit (e.g., epsilon, delta, gammaor zeta), as well as antibodies and antigen-binding fragments thereofthat specifically recognize a dimeric complex of two CD3 subunits (e.g.,gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers). The antibodiesand antigen-binding fragments of the present invention may bind solubleCD3 and/or cell surface expressed CD3. Soluble CD3 includes natural CD3proteins as well as recombinant CD3 protein variants such as, e.g.,monomeric and dimeric CD3 constructs, that lack a transmembrane domainor are otherwise unassociated with a cell membrane.

The term “conservative,” when used to describe a conservative amino acidsubstitution, includes substitution of an amino acid residue by anotheramino acid residue having a side chain R group with similar chemicalproperties (e.g., charge or hydrophobicity). Conservative amino acidsubstitutions may be achieved by modifying a nucleotide sequence so asto introduce a nucleotide change that will encode the conservativesubstitution. In general, a conservative amino acid substitution willnot substantially change the functional properties of interest of aprotein, for example, the ability of CD3 proteins to play a role in Tcell receptor assembly and signaling. Examples of groups of amino acidsthat have side chains with similar chemical properties include aliphaticside chains such as glycine, alanine, valine, leucine, and isoleucine;aliphatic-hydroxyl side chains such as serine and threonine;amide-containing side chains such as asparagine and glutamine; aromaticside chains such as phenylalanine, tyrosine, and tryptophan; basic sidechains such as lysine, arginine, and histidine; acidic side chains suchas aspartic acid and glutamic acid; and, sulfur-containing side chainssuch as cysteine and methionine. Conservative amino acids substitutiongroups include, for example, valine/leucine/isoleucine,phenylalanine/tyrosine, lysine/arginine, alanine/valine,glutamate/aspartate, and asparagine/glutamine. In some embodiments, aconservative amino acid substitution can be a substitution of any nativeresidue in a protein with alanine, as used in, for example, alaninescanning mutagenesis. In some embodiments, a conservative substitutionis made that has a positive value in the PAM250 log-likelihood matrixdisclosed in Gonnet et al. ((1992) Exhaustive Matching of the EntireProtein Sequence Database, Science 256:1443-45), hereby incorporated byreference. In some embodiments, the substitution is a moderatelyconservative substitution wherein the substitution has a nonnegativevalue in the PAM250 log-likelihood matrix.

Thus, encompassed by the invention is a genetically modified non-humananimal, e.g., rodent, e.g., mouse or rat, expressing a humanized CD3protein(s) comprising conservative amino acid substitutions in the aminoacid sequence described herein.

One skilled in the art would understand that in addition to the nucleicacid residues encoding humanized CD3 proteins described herein, due tothe degeneracy of the genetic code, other nucleic acids may encode thepolypeptides of the invention. Therefore, in addition to a geneticallymodified non-human animal that comprises in its genome nucleotidesequences encoding humanized CD3 proteins described herein, a non-humananimal that comprises in its genome nucleotide sequences that differfrom those described herein due to the degeneracy of the genetic codeare also provided.

The term “identity” when used in connection with sequence includesidentity as determined by a number of different algorithms known in theart that can be used to measure nucleotide and/or amino acid sequenceidentity. In some embodiments described herein, identities aredetermined using a ClustalW v. 1.83 (slow) alignment employing an opengap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnetsimilarity matrix (MacVector™ 10.0.2, MacVector Inc., 2008). The lengthof the sequences compared with respect to identity of sequences willdepend upon the particular sequences. In various embodiments, identityis determined by comparing the sequence of a mature protein from itsN-terminal to its C-terminal. In various embodiments, when comparing ahumanized sequence to a human sequence, the human portion of thehumanized sequence (but not the non-human portion) is used in making acomparison for the purpose of ascertaining a level of identity between ahuman sequence and a humanized sequence.

The term “operably linked” includes a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. As such, a nucleic acid sequenceencoding a protein may be operably linked to regulatory sequences (e.g.,promoter, enhancer, silencer sequence, etc.) so as to retain propertranscriptional regulation. In addition, various portions of thehumanized protein of the invention may be operably linked to retainproper folding, processing, targeting, expression, and other functionalproperties of the protein in the cell. Unless stated otherwise, variousdomains of the humanized protein of the invention are operably linked toeach other.

The term “replacement” in reference to gene replacement includes placingexogenous genetic material at an endogenous genetic locus, therebyreplacing all or a portion of the endogenous gene with an orthologous orhomologous nucleic acid sequence. In one instance, an endogenousnon-human gene or fragment thereof is replaced with a correspondinghuman gene or fragment thereof. A corresponding human gene or fragmentthereof is a human gene or fragment that is an ortholog of, a homologof, or is substantially identical or the same in structure and/orfunction, as the endogenous non-human gene or fragment thereof that isreplaced. As demonstrated in the Examples below, nucleotide sequencesencoding endogenous non-human CD3 extracellular domains were replaced bynucleotide sequences corresponding to human CD3 extracellular domains.

“Functional” as used herein, e.g., in reference to a functional protein,includes a protein that retains at least one biological activitynormally associated with the native protein. For example, in someembodiments of the invention, a replacement at an endogenous locus(e.g., replacement at endogenous non-human CD3 loci) results in a locusthat fails to express a functional endogenous protein.

The term “locus” as in CD3 locus includes the genomic DNA comprising theCD3 coding region. Other sequences may be included in the CD3 locus thathave been introduced for the purposes of genetic manipulation, e.g.,selection cassettes, restriction sites, etc.

The term “germline” in reference to an immunoglobulin nucleic acidsequence includes a nucleic acid sequence that can be passed to progeny.

The phrase “immunoglobulin molecule” includes two immunoglobulin heavychains and two immunoglobulin light chains. The heavy chains may beidentical or different, and the light chains may be identical ordifferent.

The term “antibody”, as used herein, includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chaincomprises a heavy chain variable domain and a heavy chain constantregion (C_(H)). The heavy chain constant region comprises three domains,C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a light chainvariable domain and a light chain constant region (C_(L)). The heavychain and light chain variable domains can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each heavy and light chain variable domaincomprises three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 andHCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3).

The term “high affinity” antibody refers to an antibody that has a K_(D)with respect to its target epitope about of 10⁻⁹ M or lower (e.g., about1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, or about 1×10⁻¹² M).

The phrase “bispecific antibody” includes an antibody capable ofselectively binding two epitopes. Bispecific antibodies generallycomprise two arms, each binding a different epitope (e.g., two heavychains with different specificies)—either on two different molecules(e.g., different epitopes on two different immunogens) or on the samemolecule (e.g., different epitopes on the same immunogen). If abispecific antibody is capable of selectively binding two differentepitopes (a first epitope and a second epitope), the affinity of thefirst antibody arm for the first epitope will generally be at least oneto two or three or four or more orders of magnitude lower than theaffinity of the first antibody arm for the second epitope, and viceversa. Epitopes specifically bound by the bispecific antibody can be onthe same or a different target (e.g., on the same or a differentprotein). Exemplary bispecific antibodies include those with a firstantibody arm specific for a tumor antigen and a second antibody armspecific for a cytotoxic marker, e.g., an Fc receptor (e.g., FcγRI,FcγRII, FcγRIII, etc.) or a T cell marker (e.g., CD3, CD28, etc.). Inone embodiment of the present invention, one arm of the bispecificantibody is specific for CD3. Further, a bispecific antibody with afirst arm specific for a tumor antigen and a second arm specific for atoxin can be paired so as to deliver a toxin (e.g., saporin, vincaalkaloid, etc.) to a tumor cell. Other exemplary bispecific antibodiesinclude those with a first arm specific for an activating receptor(e.g., B cell receptor, FcγRI, FcγRIIA, FcγRIIIA, FcγRI, T cellreceptor, etc.) and a second arm specific for an inhibitory receptor(e.g., FcγRIIB, CD5, CD22, CD72, CD300a, etc.). Such bispecificantibodies can be constructed for therapeutic conditions associated withcell activation (e.g., allergy and asthma). Bispecific antibodies can bemade, for example, by combining heavy chains that recognize differentepitopes of the same immunogen. For example, nucleic acid sequencesencoding heavy chain variable sequences that recognize differentepitopes of the same immunogen can be fused to nucleic acid sequencesencoding the same or different heavy chain constant regions, and suchsequences can be expressed in a cell that expresses an immunoglobulinlight chain. A typical bispecific antibody has two heavy chains eachhaving three heavy chain CDRs, followed by (N-terminal to C-terminal) aC_(H)1 domain, a hinge, a C_(H)2 domain, and a C_(H)3 domain, and animmunoglobulin light chain that either does not confer epitope-bindingspecificity but that can associate with each heavy chain, or that canassociate with each heavy chain and that can bind one or more of theepitopes bound by the heavy chain epitope-binding regions, or that canassociate with each heavy chain and enable binding of one or both of theheavy chains to one or both epitopes. Similarly, the phrase“multispecific antibody” includes an antibody capable of selectivelybinding multiple epitopes (e.g., two, three, four epitopes).

The phrase “complementarity determining region,” or the term “CDR,”includes an amino acid sequence encoded by a nucleic acid sequence of anorganism's immunoglobulin genes that normally (i.e., in a wild-typeanimal) appears between two framework regions in a variable region of alight or a heavy chain of an immunoglobulin molecule. A CDR can beencoded by, for example, a germline sequence or a rearranged orunrearranged sequence, and, for example, by a naive or a mature B cell.A CDR can be somatically mutated (e.g., vary from a sequence encoded inan animal's germline), humanized, and/or modified with amino acidsubstitutions, additions, or deletions. In some circumstances (e.g., fora CDR3), CDRs can be encoded by two or more sequences (e.g., germlinesequences) that are not contiguous (e.g., in an unrearranged nucleicacid sequence) but are contiguous in a B cell nucleic acid sequence,e.g., as the result of splicing or connecting the sequences (e.g., V-D-Jrecombination to form a heavy chain CDR3).

The phrase “functional fragment” includes fragments of antigen-bindingproteins such as antibodies that can be expressed, secreted, andspecifically bind to an epitope with a K_(D) in the micromolar,nanomolar, or picomolar range. Specific recognition includes having aK_(D) that is at least in the micromolar range, the nanomolar range, orthe picomolar range.

The phrase “heavy chain,” or “immunoglobulin heavy chain” includes animmunoglobulin heavy chain sequence, including immunoglobulin heavychain constant region sequence, from any organism. Heavy chain variabledomains include three heavy chain CDRs and four FR regions, unlessotherwise specified. Fragments of heavy chains include CDRs, CDRs andFRs, and combinations thereof. A typical heavy chain has, following thevariable domain (from N-terminal to C-terminal), a C_(H)1 domain, ahinge, a C_(H)2 domain, and a C_(H)3 domain. A functional fragment of aheavy chain includes a fragment that is capable of specificallyrecognizing an epitope (e.g., recognizing the epitope with a K_(D) inthe micromolar, nanomolar, or picomolar range), that is capable ofexpressing and secreting from a cell, and that comprises at least oneCDR. A heavy chain variable domain is encoded by a variable region genesequence, which generally comprises V_(H), D_(H), and J_(H) segmentsderived from a repertoire of V_(H), D_(H), and J_(H) segments present inthe germline. Sequences, locations and nomenclature for V, D, and Jheavy chain segments for various organisms can be found on the websitefor the International Immunogenetics Information System (IMGT database).

The phrase “light chain” includes an immunoglobulin light chain sequencefrom any organism, and unless otherwise specified includes human kappaand lambda light chains and a VpreB, as well as surrogate light chains.Light chain variable domains typically include three light chain CDRsand four framework (FR) regions, unless otherwise specified. Generally,a full-length light chain includes, from amino terminus to carboxylterminus, a variable domain that includesFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region. Alight chain variable domain is encoded by a light chain variable regiongene sequence, which generally comprises V_(L) and J_(L) segments,derived from a repertoire of V and J segments present in the germline.Sequences, locations and nomenclature for V and J light chain segmentsfor various organisms can be found on the website for the InternationalImmunogenetics Information System (IMGT database). Light chains includethose, e.g., that do not selectively bind any epitopes recognized byantigen-binding protein (e.g., antibody) in which they appear. Lightchains also include those that bind and recognize, or assist the heavychain with binding and recognizing, one or more epitopes selectivelybound by the antigen-binding protein (e.g., an antibody) in which theyappear.

The term “antigen-binding protein” as used herein includes antibodiesand various naturally produced and engineered molecules capable ofbinding the antigen of interest. Such include, e.g., domain-specificantibodies, single domain antibodies (e.g., derived from camelids andfish, etc.), domain-deleted antibodies, chimeric antibodies, CDR-graftedantibodies, diabodies, triabodies, tetrabodies, minibodies, nanabodies(e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), shark variable IgNAR domains, etc.Antigen-binding protein may also include antigen-binding fragments suchas, e.g., (i) Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments;(iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAbfragments; and (vii) minimal recognition units consisting of the aminoacid residues that mimic the hypervariable region of an antibody (e.g.,an isolated complementarity determining region (CDR) such as a CDR3peptide), etc.

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of prokaryotesand eukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In some embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In some embodiments,the cell is eukaryotic and is selected from the following cells: CHO(e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell,Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK),HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21),Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell,SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myelomacell, tumor cell, and a cell line derived from an aforementioned cell.In some embodiments, the cell comprises one or more viral genes, e.g. aretinal cell that expresses a viral gene (e.g., a PER.C6™ cell). In someembodiments, the cell is an ES cell.

Genetically Modified Humanized CD3 Animals

In various embodiments, the present invention provides geneticallymodified non-human animals (e.g., rodents, e.g., mice or rats) thatcomprise in their genome (e.g., in their germline genome) a nucleic acidsequence encoding a humanized CD3 protein (e.g., a humanized CD3ε, CD3γ,CD3δ, or combination thereof). In one embodiment, the present inventionprovides genetically modified non-human animals (e.g., rodents, e.g.,mice or rats) that comprise in their genome nucleotide sequencesencoding humanized CD3δ, humanized CD3γ, and humanized CD3ε proteins.Thus, in some embodiments of the invention, the mouse expresses ahumanized CD3γδε complex on the surface of its T cells such that thehumanized CD3γδε forms a complex with the T cell receptor expressed onthe same T cell.

CD3 molecule is commonly a target of agents that are aimed at modulatingT cell immunity, and several anti-CD3 antibodies have been developed forthat purpose (e.g., muromonab-CD3 or OKT3). Anti-CD3 antibodies such asOKT3 are used as immunosuppressive agents (e.g., in transplantrejection) but are also studied for their therapeutic potential inautoimmune diseases (e.g., Crohn's disease, type I diabetes, ulcerativecolitis, etc.).

Additionally, CD3 molecules are also being studied as targets forbispecific agents, e.g., bispecific antibodies, because of the abilityof anti-CD3 bispecific antibodies to recruit T cells to a target cell,e.g., a cell that expresses a particular antigen of interest. Exemplaryanti-CD3 bispecific antibodies are described in U.S. Patent ApplicationPublication No. 2014/0088295, and US. Patent Application No. 62/033,460(filed Aug. 5, 2014), both incorporated herein by reference.

During preclinical drug development stage, candidate agents aretypically studied based on their efficacy, toxicity, and otherpharmacokinetic and pharmacodynamics properties. Candidate agents, suchas antibodies, typically target a human antigen—as the end goal ofinvestigation is to develop a human therapy. Many preclinical studiesare conducted in large animals such as primates as their physiology anddrug metabolism are most similar to humans. Several antibodies developedto CD3 (e.g., OKT3) are known not to cross-react to non-human CD3,particularly to primate CD3. To conduct effective preclinicalinvestigations relating to efficacy, toxicity, and other parameters of adrug candidate, first, the drug candidate must be determined torecognize primate CD3 molecule.

However, a separate factor complicating development of anti-CD3 therapyis that large primates such as chimpanzees are endangered and in manycountries studies in chimpanzees are prohibited; while studies in otherprimates, e.g., cynomolgus monkeys (Macaca fascicularis), may raiseethical concerns. Thus, any preliminary data on a specific therapeuticcandidate that can be obtained in a smaller animal model, such as arodent, e.g., a mouse, can be helpful in determining further progress ofpreclinical investigations in large primates.

The most useful small animal model to conduct preliminary studies is anon-human animal, e.g., a rodent, that expresses a human or humanizedCD3 protein, and allows the testing of anti-CD3 drug candidates thatalso cross-react with cynomolgus monkey CD3, allowing for subsequentprimate preclinical studies. The present invention provides such anintricate animal model.

Thus, provided herein is a genetically modified non-human animalcomprising in its genome a nucleic acid sequence(s) encoding anextracellular domain of a human CD3 protein. In some embodiments of theinvention, the CD3 protein is selected from the group consisting ofCD3γ, CD3δ, CD3ε, CD3ζ, and a combination thereof. In some embodiments,the CD3 protein is selected from the group consisting of CD3γ, CD3δ,CD3ε, and a combination thereof. In some embodiments, the CD3 proteincomprises CD3γ, CD3δ, and CD3ε polypeptide chains. Thus, in someembodiments, the genetically modified non-human animal comprises in itsgenome a nucleic acid sequence(s) encoding an extracellular domain of ahuman a CD3γ, an extracellular domain of a human CD3δ, and anextracellular domain of a human CD3ε. In some such embodiments, theextracellular domains of human CD3γ, CD3δ, and CD3ε may be encoded by asingle nucleic acid. In some embodiments, the extracellular domains ofhuman CD3γ, CD3δ, and CD3ε are encoded by separate nucleic acids.

Exemplary CD3 proteins are presented in the alignment in FIG. 4. A mouseCD3ε protein sequence can be found in GenBank Accession Number NP_031674and SEQ ID NO:27, while a human CD3ε protein sequence can be found inGenBank Accession Number NP_000724 and SEQ ID NO:28. A mouse CD3δprotein sequence can be found in GenBank Accession Number NP_038515 andSEQ ID NO:29, while a human CDδ protein sequence can be found in GenBankAccession Number NP_000723 and SEQ ID NO:30. A mouse CD3γ proteinsequence can be found in GenBank Accession Number NP_033980 and SEQ IDNO:31, while a human CD3γ protein sequence can be found in GenBankAccession Number NP_000064 and SEQ ID NO:32.

In some embodiments of the invention, the nucleic acid sequence(s)encoding an extracellular domain of a human CD3, e.g., an extracellulardomain of a human a CD3γ, human CD3δ, and human CD3ε, are located at anendogenous non-human CD3 locus. In some embodiments of the invention,the non-human animal does not comprise a functional extracellular domainof the corresponding non-human CD3 protein. In some embodiments of theinvention, the nucleic acid sequence(s) encoding an extracellular domainof a human CD3 replaces corresponding nucleic acid sequence(s) encodingendogenous non-human CD3. Thus, in some embodiments, the nucleic acidsequence encoding the extracellular domain of a human CD3γ replaces thenucleic acid sequence encoding the extracellular domain of endogenousnon-human CD3γ, the nucleic acid sequence encoding the extracellulardomain of a human CD3δ replaces the nucleic acid sequence encoding theextracellular domain of endogenous non-human CD3δ, and the nucleic acidsequence encoding the extracellular domain of a human CD3ε replaces thenucleic acid sequence encoding the extracellular domain of endogenousnon-human CD3ε. In some embodiments, the replacement does not comprisethe replacement of a nucleic acid sequence encoding endogenous signalsequence. In another embodiment, the replacement comprises thereplacement of the nucleic acid sequence encoding endogenous signalsequence with the nucleic acid sequence encoding a human signalsequence.

In some aspects of the invention, the extracellular domain comprises theregion of the protein(s) that is not a transmembrane or a cytoplasmicdomain, e.g., the region of the protein that appears on the surface ofthe cell and that, in part, when assembled in a complex interacts withthe extracellular domains of other components of TCR signaling complex,e.g., TCR alpha and beta extracellular domains. In various embodimentsdescribed herein, extracellular domain refers to the domain of theprotein expressed on the cell surface and, unless indicated otherwise,does not include the signal sequence which is typically proteolyticallycleaved prior to sell surface expression. In some embodiments of theinvention, the extracellular domain of CD3ε comprises amino acids 17-130of the amino acid sequence set forth in SEQ ID NO:24 (set forthseparately as SEQ ID NO:33). In some such embodiments, the animalcomprises the nucleic acid sequence encoding an endogenous CD3ε signalsequence, e.g., signal sequence at amino acids 1-16 of SEQ ID NO:24. Inother embodiments of the invention, the animal comprises the nucleicacid sequence encoding a human CD3ε signal sequence. In some embodimentsof the invention, the extracellular domain of CD3δ comprises amino acids19-105 of the amino acid sequence set forth in SEQ ID NO:25 (set forthseparately as SEQ ID NO:34). In some such embodiments, the animalcomprises the nucleic acid sequence encoding an endogenous CD3δ signalsequence, e.g., signal sequence at amino acids 1-18 of SEQ ID NO:25. Inother embodiments of the invention, the animal comprises the nucleicacid sequence encoding a human CD3δ signal sequence. In someembodiments, the extracellular domain of CD3γ comprises amino acids20-116 of the amino acid sequence set forth in SEQ ID NO:26 (set forthseparately as SEQ ID NO:35). In some such embodiments, the animalcomprises the nucleic acid sequence encoding endogenous CD3γ signalsequence, e.g., signal sequence at amino acids 1-19 of SEQ ID NO:26. Inother embodiments of the invention, the animal comprises the nucleicacid sequence encoding a human CD3γ signal sequence.

In some aspects of the invention, the non-human animal comprises anucleic acid sequence encoding transmembrane and cytoplasmic domains ofendogenous CD3 protein, e.g., corresponding endogenous CD3 protein.Thus, in one embodiment, the non-human animal comprises a nucleic acidsequence encoding the extracellular domain of the human CD3 proteinoperably linked to the nucleic acid sequence encoding transmembrane andcytoplasmic domains of the corresponding endogenous non-human CD3protein. Thus, in one aspect, the animal comprises at an endogenous CD3locus a nucleic acid sequence(s) encoding an extracellular domain of ahuman CD3 protein operably linked to a nucleic acid sequence(s) encodingtransmembrane and cytoplasmic domains of an endogenous non-human CD3. Inone embodiment, the animal comprises at an endogenous CD3ε locus anucleic acid sequence encoding an extracellular domain of a human CD3εoperably linked to a nucleic acid sequence encoding transmembrane andcytoplasmic domains of an endogenous non-human animal CD3ε, at anendogenous CD3δ locus a nucleic acid sequence encoding an extracellulardomain of a human CD3δ operably linked to a nucleic acid sequenceencoding transmembrane and cytoplasmic domains of an endogenousnon-human animal CD3δ, and at an endogenous CD3γ locus a nucleic acidsequence encoding an extracellular domain of a human CD3γ operablylinked to a nucleic acid sequence encoding transmembrane and cytoplasmicdomains of an endogenous non-human animal CD3γ.

In some aspects of the invention, the non-human animal expressesextracellular domains of human CD3 protein. In some aspects, thenon-human animal expresses an extracellular domain of human CD3ε setforth in SEQ ID NO:33. In some aspects, the non-human animal expressesan extracellular domain of human CD3δ set forth in SEQ ID N0:34. In someaspects, the non-human animal expresses an extracellular domain of humanCD3γ set forth in SEQ ID NO:35.

In some embodiments of the invention, the non-human animal is a mammal.In one aspect, the non-human animal is a small mammal, e.g., of thesuperfamily Dipodoidea or Muroidea. In one embodiment, the geneticallymodified animal is a rodent. In one embodiment, the rodent is selectedfrom a mouse, a rat, and a hamster. In one embodiment, the rodent isselected from the superfamily Muroidea. In one embodiment, thegenetically modified animal is from a family selected from Calomyscidae(e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World ratsand mice, voles), Muridae (true mice and rats, gerbils, spiny mice,crested rats), Nesomyidae (climbing mice, rock mice, white-tailed rats,Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), andSpalacidae (e.g., mole rats, bamboo rats, and zokors). In a specificembodiment, the genetically modified rodent is selected from a truemouse or rat (family Muridae), a gerbil, a spiny mouse, and a crestedrat. In one embodiment, the genetically modified mouse is from a memberof the family Muridae. In one embodiment, the animal is a rodent. In aspecific embodiment, the rodent is selected from a mouse and a rat. Inone embodiment, the non-human animal is a mouse.

In one embodiment, the non-human animal is a rodent that is a mouse of aC57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN,C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn,C57BL/10Cr, and C57BL/Ola. In another embodiment, the mouse is a 129strain selected from the group consisting of a strain that is 129P1,129P2, 129P3, 129X1, 129S1 (e.g., 12951/SV, 129S1/SvIm), 129S2, 129S4,129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2(see, e.g., Festing et al. (1999) Revised nomenclature for strain 129mice, Mammalian Genome 10:836, see also, Auerbach et al (2000)Establishment and Chimera Analysis of 129/SvEv- and C57BL/6-DerivedMouse Embryonic Stem Cell Lines). In a specific embodiment, thegenetically modified mouse is a mix of an aforementioned 129 strain andan aforementioned C57BL/6 strain. In another specific embodiment, themouse is a mix of aforementioned 129 strains, or a mix of aforementionedBL/6 strains. In a specific embodiment, the 129 strain of the mix is a129S6 (129/SvEvTac) strain. In another embodiment, the mouse is a BALBstrain, e.g., BALB/c strain. In yet another embodiment, the mouse is amix of a BALB strain and another aforementioned strain.

In one embodiment, the non-human animal is a rat. In one embodiment, therat is selected from a Wistar rat, an LEA strain, a Sprague Dawleystrain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment,the rat strain is a mix of two or more strains selected from the groupconsisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and DarkAgouti.

Thus, in one embodiment, the genetically modified non-human animal is arodent. In one embodiment, the genetically modified non-human animal isa rat or a mouse. In one embodiment, the animal is a mouse. Thus, in oneembodiment, the genetically modified animal is a mouse and the mousecomprises at an endogenous mouse CD3 locus a nucleotide sequenceencoding an extracellular domain of a human CD3 protein. In oneembodiment, the mouse comprises a nucleic acid sequence encoding anextracellular domain of a human CD3ε, a nucleic acid sequence encodingan extracellular domain of a human CD3δ, and a nucleic acid sequenceencoding an extracellular domain of a human CD3γ. In some embodiments ofthe invention, the extracellular domain of the human CD3ε comprises thesequence set forth in SEQ ID NO:33, the extracellular domain of thehuman CD3δ comprises the sequence set forth in SEQ ID NO:34, and theextracellular domain of the human CD3γ comprises the sequence set forthin SEQ ID NO:35. In some embodiments, the mouse comprises thesequence(s) encoding endogenous mouse CD3 signal sequence(s). In otherembodiments, the mouse comprises the sequence(s) encoding human CD3signal sequence(s).

In some embodiments of the invention, the mouse of the inventionexpresses humanized CD3 protein(s). In one embodiment, the mouseexpresses humanized CD3ε, humanized CD3δ, and humanized CD3γ proteins.In some embodiments of the invention, the mouse expresses a human CD3εextracellular domain and endogenous mouse CD3ε transmembrane andcytoplasmic domains, a human CD3δ extracellular domain and endogenousmouse CD3δ transmembrane and cytoplasmic domains, and a human CD3γextracellular domain and endogenous mouse CD3γ transmembrane andcytoplasmic domains. In some such embodiments, the mouse expresseshumanized CD3 proteins wherein the humanized CD3 proteins are humanizedCD3ε set forth in SEQ ID NO:24, humanized CD3δ set forth in SEQ IDNO:25, and humanized CD3γ set forth in SEQ ID NO:26.

In some aspects of the invention, the genetically engineered mouse is animmunocompetent mouse. In some embodiments of the invention, theintroduction of humanized CD3 protein(s) does not affect the mouse'simmune system function. In some embodiments of the invention, the mousecomprises normal T and B cell ratio. In some embodiments of theinvention, the mouse is capable of mounting a normal response to mouseinfection. In some aspects, the mouse displays similar CD4+ to CD8+ cellratio in the thymus as compared to a wild type mouse, e.g., a mouse thathas not been genetically modified to express humanized CD3 protein(s).In some embodiments of the invention, the CD4+ to CD8+ cell ratio in thethymus of the mouse is within 30%, e.g., within 20%, e.g., within 15%,e.g., within 12%, e.g., within 10%, e.g., within 5%, e.g., within 2%, ofthe CD4+ to CD8+ cell ratio of a mouse that is not genetically modifiedto express humanized CD3 protein(s). In some aspects, the mouse displayssimilar T and B cell percentages in the spleen, lymph nodes, andperipheral blood as a wild type mouse, e.g., a mouse that is notgenetically modified to express humanized CD3 protein(s). In someaspects, the mouse displays similar numbers of circulating white bloodcells, lymphocytes, monocytes, neutrophils, eosinophils, and basophilsas a wild type mouse, e.g., a mouse that is not genetically modified toexpress humanized CD3 protein(s).

Also provided herein are methods of making the genetically modifiednon-human animal described herein. In some embodiments, the method ofmaking a genetically modified non-human animal wherein the animalexpresses a humanized CD3 protein comprises placing at an endogenousnon-human animal CD3 locus a nucleic acid sequence encoding anextracellular domain of a human CD3 protein, wherein the human CD3protein is selected from the group consisting of CD3ε, CD3δ, CD3γ, CD3ζ,and a combination thereof. In one embodiment the animal does notcomprise a functional extracellular domain of the correspondingnon-human CD3 protein(s). In one aspect, the animal comprises at anendogenous non-human CD3 locus a nucleic acid sequence encoding anextracellular domain of human CD3ε, an extracellular domain of humanCD3δ, and an extracellular domain of human CD3γ. In some embodiments,the extracellular domain of a human CD3ε is set forth in SEQ ID NO:33,the extracellular domain of a human CD3δ is set forth in SEQ ID NO: 34,and the extracellular domain of a human CD3γ is set forth in SEQ IDNO:35. In one embodiment, the animal does not comprise a functionalextracellular domain of the corresponding non-human CD3 protein(s).

In some embodiments, the method of making a genetically modifiednon-human animal of the invention comprises replacing at the endogenousCD3 locus a nucleotide sequence encoding the extracellular domain of anon-human CD3 protein(s) with a nucleotide sequence encoding anextracellular domain of a corresponding human CD3 protein(s). In oneembodiment, the animal retains transmembrane and cytoplasmic domains ofthe non-human CD3 protein(s). In some embodiments, the replacementresults in a chimeric protein(s) comprising an extracellular domain of ahuman CD3 protein(s) and transmembrane and cytoplasmic domains ofcorresponding endogenous non-human CD3 protein(s).

In some embodiments, the replacement method utilizes a targetingconstruct made using VELOCIGENE® technology, introducing the constructinto ES cells, and introducing targeted ES cell clones into a mouseembryo using VELOCIMOUSE® technology, as described in the Examples.

In one embodiment, wherein the method comprises the replacement of thenucleotide sequence encoding the extracellular domain of endogenousnon-human CD3ε with the nucleotide sequence encoding the extracellulardomain of a human CD3ε protein, the method comprises a replacement ofpartial sequence of endogenous mouse coding exons 2 to 4 of mouse CD3εgene with partial sequence of human coding exons 2 to 5 of human CD3εgene. In one embodiment, wherein the method comprises the replacement ofthe nucleotide sequence encoding the extracellular domain of endogenousnon-human CD3δ with the nucleotide sequence encoding the extracellulardomain of a human CD3δ, the method comprises a replacement of partialsequence of endogenous mouse coding exons 2 to 3 of mouse CD3δ with thepartial sequence of human coding exons 2 to 3 of human CD3δ gene. In oneembodiment, wherein the method comprises a replacement of the nucleotidesequence encoding the extracellular domain of endogenous non-human CD3γwith the nucleotide sequence encoding the extracellular domain of humanCD3γ, the method comprises replacement of partial sequence of mousecoding exons 2 to 4 of mouse CD3γ with the partial sequence of humancoding exons 2 to 4 of human CD3γ gene. In one embodiment of theinvention, the replacement comprises the replacement of sequence ofCD3ε, CD3δ, and CD3γ. In such an embodiment, the replacement may beaccomplished by creating a large targeting vector that incorporates thesequential genetic modification in all three loci and then introducingthe large targeting vector into mouse ES cells to make a mouse, e.g., asdescribed in Example 1.

Thus, in one embodiment, provided herein is a large targeting vector formaking a genetically modified animal of the invention. In oneembodiment, the large targeting vector comprises 5′ and 3′ mousehomology arms; a DNA fragment comprising the CD3ε gene which comprises areplacement of partial sequence of mouse CD3ε coding exons 2 to 4 withpartial sequence of human CD3ε coding exons 2 to 5; a DNA fragmentcomprising the CD3δ gene which comprises a replacement of partialsequence of mouse CD3δ coding exons 2 to 3 with partial sequence ofhuman CD3δ coding exons 2 to 3; a DNA fragment comprising the CD3γ genewhich comprises a replacement of partial sequence of mouse CD3γ codingexons 2 to 4 with partial sequence of human CD3γ coding exons 2 to 4;and a selection cassette.

A selection cassette is a nucleotide sequence inserted into a targetingconstruct to facilitate selection of cells (e.g., bacterial cells, EScells) that have integrated the construct of interest. A number ofsuitable selection cassettes are known in the art (Neo, Hyg, Pur, CM,SPEC, etc.). In addition, a selection cassette may be flanked byrecombination sites, which allow deletion of the selection cassette upontreatment with recombinase enzymes. Commonly used recombination sitesare loxP and Frt, recognized by Cre and Flp enzymes, respectively, butothers are known in the art. A selection cassette may be locatedanywhere in the construct outside the coding region. In one embodiment,the selection cassette is inserted upstream of human CD3ε insertedsequence.

Upon completion of gene targeting, ES cells or genetically modifiednon-human animals are screened to confirm successful incorporation ofexogenous nucleotide sequence of interest or expression of exogenouspolypeptide. Numerous techniques are known to those skilled in the art,and include (but are not limited to) Southern blotting, long PCR,quantitative PCR (e.g., real-time PCR using TAQMAN®), fluorescence insitu hybridization, Northern blotting, flow cytometry, Western analysis,immunocytochemistry, immunohistochemistry, etc. In one example,non-human animals (e.g., mice) bearing the genetic modification ofinterest can be identified by screening for loss of mouse allele and/orgain of human allele using a modification of allele assay described inValenzuela et al. (2003) High-throughput engineering of the mouse genomecoupled with high-resolution expression analysis, Nature Biotech.21(6):652-659. Other assays that identify a specific nucleotide or aminoacid sequence in the genetically modified animals are known to thoseskilled in the art.

In one aspect, a method for making a chimeric human/non-human CD3molecule is provided, comprising expressing in a single cell a chimericCD3 protein from a nucleotide construct as described herein. In oneembodiment, the nucleotide construct is a viral vector; in a specificembodiment, the viral vector is a lentiviral vector. In one embodiment,the cell is selected from a CHO, COS, 293, HeLa, and a retinal cellexpressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

In one aspect, a cell that expresses a chimeric human/non-human CD3protein is provided. In one embodiment, the cell comprises an expressionvector comprising a chimeric CD3 sequence as described herein. In oneembodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinalcell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

A chimeric CD3 molecule made by a non-human animal as described hereinis also provided, wherein, in one embodiment, the chimeric CD3 moleculecomprises an amino acid sequence of all or substantially all of anextracellular domain of a human CD3 protein, and at least transmembraneand cytoplasmic domains from a non-human CD3 protein, e.g., mouse CD3protein.

In addition to a genetically engineered non-human animal, a non-humanembryo (e.g., a rodent, e.g., a mouse or a rat embryo) is also provided,wherein the embryo comprises a donor ES cell that is derived from anon-human animal (e.g., a rodent, e.g., a mouse or a rat) as describedherein. In one aspect, the embryo comprises an ES donor cell thatcomprises the chimeric CD3 gene, and host embryo cells.

Also provided is a tissue, wherein the tissue is derived from anon-human animal (e.g., a rodent, e.g., a mouse or a rat) as describedherein, and expresses the chimeric CD3 protein.

In addition, a non-human cell isolated from a non-human animal asdescribed herein is provided. In one embodiment, the cell is an ES cell.In one embodiment, the cell is a T cell. In one embodiment, the cell isa CD8+ T cell. In another embodiment, the cell is a CD4+ T cell.

In some embodiments, also provided herein are genetic loci comprisingthe nucleic acid sequences that encoding the humanized CD3 protein(s)described herein.

Mouse Model for Testing Human Therapies

In some aspects, provided herein is a mouse model for testingCD3-targeted (“anti-CD3”) therapeutic agents. In some embodiments,provided herein is a mouse model for testing anti-CD3 antigen-bindingproteins. In some embodiments, provided herein is a mouse model fortesting anti-CD3 antibodies. In some such embodiments, provided is amouse model for testing anti-CD3 multispecific, e.g. bispecificantigen-binding proteins or anti-CD3 bispecific antibodies. As such, ananti-CD3 multispecific antigen-binding protein, e.g. an anti-CD3bispecific antigen-binding protein, targets or specifically binds saidhumanized CD3 protein and at least one other antigen of interest. Invarious aspects, the mouse model for testing anti-CD3 bispecificantigen-binding proteins wherein the antigen-binding protein is capableof binding both CD3 and the antigen of interest comprises a nucleic acidsequence encoding a humanized CD3 protein, wherein the humanized CD3protein is selected from the group consisting of CD3ε, CD3δ, CD3γ, CD3ζ,and a combination thereof, and a cell expressing or comprising theantigen of interest. In one embodiment, the mouse comprises a T cellexpressing said humanized CD3 protein(s).

In an embodiment, the testing of the monospecific or bispecificantigen-binding protein involves performing an assay or a study thatallows determination of the effect of the antigen-binding protein on theT cell expressing said humanized CD3 protein. In another embodiment, thetesting of the bispecific antigen-binding protein involves performing anassay or a study that allows determination of the effect of theantigen-binding protein on both the T cell expressing said humanized CD3protein and the cell expressing or comprising the antigen of interest,or the interaction between said CD3-expressing T cell and the cellexpressing or comprising the antigen of interest. In one embodiment, thetesting of the monospecific or bispecific antigen-binding proteininvolves performing an assay or a study that allows determination of theeffect of the T cell expressing said humanized CD3 protein on the cellexpressing or comprising said antigen of interest. In one embodiment,such assay measures, e.g., the number of cells expressing the antigen ofinterest, immune response, cellular interactions, cellular cytotoxicity,cytokine release, cellular activation, cell proliferation, tumor growthor regression, changes in pathology, or the like. Various assays includebut are not limited to measurements of complement-directed cytotoxicity(CDC), antibody-dependent cytotoxicity (ADCC), antibody-dependentcellular phagocytosis (ADCP), PBMC proliferation, CD69 activation,histological tissue analysis, analysis of tissue and cellular biomarkers(e.g., cells or tissue may be extracted from the mouse for the purposeof the assays, or analyzed by radiography, MRI, PET, SPECT, BLI, andfluorescence-based imaging modalities).

In some embodiments of the invention, in such a mouse model, the antigenof interest has been introduced into said mouse. The antigen of interestmay be introduced by several methods known to those skilled in the art.Some nonlimiting methods include transgenesis, injection, infection,tissue or cell transplantation. The antigen of interest or a fragmentthereof (e.g., a fragment that is recognized by the antigen-bindingprotein being tested) can be targeted to, or expressed by, particularcell types. In some embodiments, the antigen of interest is a humanizedantigen of interest encoded by the mouse genome.

The antigen of interest may be a membrane-bound protein such that it isexpressed only on cell surface. Alternatively, the antigen of interestor a fragment thereof (e.g., a fragment that is recognized by theantigen-binding protein being tested) may be displayed on the cellsurface complexed with another protein or moiety. Some cell-surfaceantigens may associate with other proteins as co-receptor complexes, orbind or have affinity to extracellular molecules. Thus, the mouse modelmay be utilized to test bispecific antigen-binding molecules thatinteract with T cells in various cell systems.

In one embodiment, the mouse model expresses human CD3ε, CD3δ, CD3γextracellular domains. In one embodiment, the mouse expresses mousetransmembrane and cytoplasmic domain of CD3ε, CD3δ, and CD3γ; an in oneembodiment, the transmembrane and cytoplasmic domains are endogenousmouse domains. In one embodiment, the mouse model expresses CD3ε, CD3δ,and CD3γ, each comprising a human extracellular domain and mouse, e.g.,endogenous mouse, transmembrane and cytoplasmic domains.

In various embodiment of the invention, the antigen-binding proteinbinds both CD3 and the antigen of interest in the mouse model. In oneembodiment, the antigen of interest is a human antigen. In oneembodiment, the antigen of interest is a primate antigen, e.g., acynomolgus monkey antigen. In one embodiment, the antigen-bindingprotein is capable of binding the same antigen of interest of both humanand monkey origin. In one embodiment, the antigen-binding protein iscapable of binding both human and monkey CD3.

In one embodiment, the mouse model comprises a xenograft of a tumorexpressing the antigen of interest. In one embodiment, the cellexpressing or comprising the antigen of interest in said mouse is animmortalized cell, such as a tumor cell. Thus, the mouse model isutilized to test the activity of anti-CD3 bispecific antigen-bindingproteins in blocking or affecting the tumor cell expressing the antigenof interest.

Thus, in the embodiment of the invention, wherein the cell expressing orcomprising the antigen of interest is a tumor cell, the antigen ofinterest may be a tumor-associated antigen (TAA). Various tumor antigensare listed in the database of T cell defined tumor antigens (van derBruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptide database: Tcell-defined tumor antigens. Cancer Immun 2013). Exemplary tumorassociated antigens include but are not limited to AFP, ALK, BAGEproteins, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX,caspase-8, CCR5, CD19, CD20, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1,CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM,EphA2, Fra-1, FOLR1, GAGE protein, GD2, GD3, GloboH, glypican-3, GM3,gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGEprotein, MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16(CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53,PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGEproteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, survivin,TAG-72, TGF-β, TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase, and uroplakin-3.

In another embodiment of the invention, the mouse model is used todetermine if a candidate bispecific antigen-binding protein is capableof blocking or affecting an antigen of interest which is an infectiousdisease associated antigen. In one embodiment of the invention, themouse is infected with an infectious agent. In one embodiment of theinvention, the infectious disease associated antigen is a viral antigen.In one aspect, the viral antigen is selected from the group consistingof HIV, hepatitis A, hepatitis B, hepatitis C, herpes virus (e.g.,HSV-1, HSV-2, CMV, HAV-6, VZV, Epstein Barr virus), adenovirus,influenza virus, flavivirus, echovirus, rhinovirus, coxsackie virus,coronavirus, respiratory syncytial virus, mumps virus, rotavirus,measles virus, rubella virus, parvovirus, vaccinia virus, HTLV, denguevirus, papillomavirus, molluscum virus, poliovirus, rabies virus, JCvirus, ebola virus, and arboviral encephalitis virus antigen.

In another embodiment of the invention, wherein the antigen of interestis an infectious disease associated antigen, the antigen of interest isa bacterial antigen. In some aspects of the invention, the bacterialantigen is selected from the group consisting of chlamydia, rickettsia,mycobacteria, staphylococci, streptococci, pneumonococci, meningococci,gonococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospira, and Lyme disease bacterial antigen.

In some aspects of the invention, the CD3-based bispecific antigenbinding protein is a human CD3 based antigen binding protein. In oneembodiment, the antigen binding protein is an antibody, e.g., a humanantibody, or an antigen-binding fragment thereof.

In some embodiments of the invention, the mouse model is animmunocompetent mouse model. In some embodiments of the invention, themouse model allows for testing of efficacy and/or toxicity of anantigen-binding protein of interest. The measures of efficacy willdepend on the antigen of interest being targeted by the bispecificagent. In some embodiments, the measure of efficacy is T cell killing ofthe cell expressing the antigen. In other embodiments, the measure ofefficacy is neutralization of the virus. In other embodiment, themeasure of efficacy may be viability of the animal. In yet anotherembodiment, the measure of efficacy may be elimination of cellsexpressing the antigen of interest, proliferation of T cells, productionof cytokines (e.g., IFNg, TNFa, IL-1, IL-2, IL-10, IL4, IL-6, granzyme,perforin, etc.)

In some embodiments of the invention, the toxicity in the animal may bemeasured as an adverse event in the animal, e.g., change in body weight,appetite, digestive changes, changes in blood cell counts, splenomegaly,histological changes of the organs, change in liver enzyme function,changes in urinalysis, organ toxicity, hemorrhage, dehydration, loss offur and scruffiness, or other signs of morbidity. One measure may bedetermination of antigen-binding protein cross-reactivity withirrelevant antigens, which, in one embodiment, can be detected by organhistology, specifically detection of antigen-binding protein in tissuesor cell types that are not known to express the antigen of interest.

Use of Genetically Modified Non-Human Animals

The invention also provides various methods of using the geneticallymodified non-human animals described herein.

In one embodiment, provided herein is a method of screening therapeuticdrug candidates that target an antigen of interest comprising (a)providing or receiving a genetically modified mouse comprising at itsendogenous mouse CD3 locus a nucleic acid sequence encoding anextracellular domain of a human CD3 protein selected from the groupconsisting of CD3ε, CD3δ, CD3γ, CD3ζ, and a combination thereof, (b)introducing into said genetically modified mouse an antigen of interest,(c) contacting said mouse with a drug candidate of interest, wherein thedrug candidate is directed against the human CD3 and the antigen ofinterest, and (d) determining if the drug candidate is efficacious inpreventing, reducing or eliminating cells characterized by the presenceor expression of the antigen of interest. In various embodiments, themouse expresses a functional humanized CD3 protein on the surface of itsT cells. In one embodiment of the method, the genetically modified mousecomprises at the endogenous mouse CD3 locus a nucleic acid sequenceencoding an extracellular domain of human CD3ε, an extracellular domainof human CD3δ, and an extracellular domain of human CD3γ. In oneembodiment of the method described herein, the mouse does not comprisethe nucleic acid sequence encoding a functional extracellular domain ofthe corresponding mouse protein. In some embodiments of the method, theextracellular domain(s) of the human CD3 protein(s) is operably linkedto the transmembrane and cytoplasmic domain(s) of the correspondingendogenous mouse CD3 protein(s). In various such embodiments of themethods, the extracellular domain of a human CD3ε is set forth in SEQ IDNO:33, the extracellular domain of a human CD3δ is set forth in SEQ IDNO:34, and the extracellular domain of a human CD3γ is set forth as SEQID NO:35. In various embodiment of the methods described here, the mousemay express a humanized CD3ε protein set forth in SEQ ID NO:24, ahumanized CD3δ protein set forth in SEQ ID NO:25, and a humanized CD3γset forth in SEQ ID N0:26.

In various embodiments of the method described herein, introduction ofthe antigen of interest into the genetically modified mouse describedherein may be accomplished by any methods known to those skilled in theart, which may include, without limitation, transgenesis, injection,infection, tissue or cell transplantation. As such, introduction may beachieved by expressing in the mouse the antigen of interest, which cancomprise genetically modifying said mouse to express the antigen ofinterest. Alternatively, introduction may comprise introduction intosaid mouse a cell expressing the antigen of interest, e.g., as in cellor tissue transplantation. Introduction may also comprise infecting saidmouse with the antigen of interest, e.g., as in bacterial or viralinfection. In one embodiment, the antigen of interest may be a humanantigen of interest. In another embodiment, it may be a bacterial or aviral antigen of interest.

The antigen of interest may be a tumor-associated antigen, as describedin detail above. The antigen may also be an infectious diseaseassociated antigen, e.g., a bacterial or a viral antigen, as describedin detail above.

In various embodiments of the methods of screening a therapeutic drugcandidate, the drug candidate may be an antigen-binding protein, e.g.,an antibody, e.g., a bispecific antibody. In various aspects, such drugcandidate is capable of binding both human CD3 and the antigen ofinterest. The antigen of interest may be a human antigen. The antigen ofinterest may also be a primate, e.g., a monkey, antigen. Thus, the drugcandidate used for screening may be capable of binding both a humanantigen and a corresponding primate antigen, in addition to bindinghuman CD3. The drug candidate may also be capable of binding primate,e.g., monkey, CD3. Thus, the drug candidate may be capable of bindingboth human and primate, e.g., monkey, CD3; and also, in one embodiment,be capable of binding a human antigen of interest. In anotherembodiment, the antigen of interest may be a bacterial or a viralantigen, and the drug candidate may be capable of binding both the humanand primate, e.g., monkey, CD3 and the bacterial or viral antigen ofinterest.

In some aspects, the therapeutic candidate is an antibody, which is ahuman antibody. In other aspects, it may be a humanized antibody. Forexample, the therapeutic candidate may be an antibody generated inVELOCIMMUNE® mice (U.S. Pat. No. 8,502,018, incorporated herein byreference); thus, the initial antibody candidate may comprise a humanvariable region and a mouse constant region. The mouse constant regionof the antibody candidate may be reengineered to be of human origin byexpressing the human variable region selected in VELOCIMMUNE® mice inoperable linkage with a human constant region.

In various embodiments of the methods described herein, the therapeuticcandidate is capable of reducing, eliminating, or preventing a disease.In one embodiment, the disease is a tumor, and the therapeutic candidateis capable of reducing, eliminating, or preventing tumor growth ascompared to an agent that does not target the antigen of interest. Insuch an embodiment of the method, determination whether the drugcandidate is efficacious in preventing, reducing or eliminating cellscharacterized by the presence or expression of the antigen of interestcan be performed using a tumor volume assay, a tumor cell killing assay,induction of apoptotic markers in tumors, reduction in blood vesselgrowth in tumors, infiltration of immune cells into tumors, etc. Inanother embodiment, the disease is an infectious disease, and atherapeutic candidate is capable reducing, eliminating, or preventing abacterial or a viral infection as compared to an agent that does nottarget the antigen of interest. In such an embodiment of the method,determination whether the drug candidate is efficacious in preventing,reducing or eliminating cells characterized by the presence orexpression of the antigen of interest can be performed using a measureof bacterial or viral titers, induction of apoptotic markers in infectedcells, etc.

Other methods of use of the humanized CD3 mice of the present inventionare also provided. In various embodiments of the invention, thehumanized CD3 mouse is an immunocompetent mouse. For example, thehumanized CD3 mouse of the invention, which comprises a healthy normalimmune system with intact development and complete complement of allimmune cell types and intact immune signaling pathways, can be used tostudy the effects of various therapeutic candidates on specific celltypes, cytokines, chemokines, etc. The mouse can then be used to answermechanistic questions relating to drug candidate function.

In addition, the humanized CD3 mice can be used in methods that involvetesting the effect of bispecific anti-CD3 drug candidates on tumorgrafts. Previously developed mouse models were immunocompromised mousemodels to allow for proper human tumor engraftment. Humanized CD3 mouseis fully immunocompetent and allows introduction and growth of tumorcells expressing the antigen of interest, so full affect on the immuneresponse can be studied, included but not limited to answeringmechanistic questions, early toxicity questions, early efficacyquestions, etc.

EXAMPLES

The following examples are provided so as to describe to those ofordinary skill in the art how to make and use methods and compositionsof the invention, and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature, etc.)but some experimental errors and deviations should be accounted for. TheExamples do not include detailed descriptions of conventional methodsthat would be well known to those of ordinary skill in the art(molecular cloning techniques, etc.). Unless indicated otherwise, partsare parts by weight, molecular weight is average molecular weight,temperature is indicated in Celsius, and pressure is at or nearatmospheric.

Example 1. Construction of Humanized CD3 Locus Example 1.1. Constructionof Humanized CD3γδε

The mouse CD3 locus was humanized by construction of unique targetingvectors from human and mouse bacterial artificial chromosomes (BAC) DNAusing VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 andValenzuela et al. (2003) High-throughput engineering of the mouse genomecouple with high-resolution expression analysis. Nat. Biotech. 21(6):652-659, both incorporated herein by reference). DNA from mouse BACbMQ-425K11 was modified by homologous recombination to replace thegenomic DNA encoding portions of mouse CD3ε, CD3δ, and CD3γ genes (mouseCD3 genes located within close proximity to one another on chromosome 9)with corresponding portions of CD3ε, CD3δ, and CD3γ genes derived fromhuman BAC RP11-414G21 (human CD3 genes are located within closeproximity to one another on chromosome 11), respectively.

Specifically, to generate humanized CD3γδε mice, the mouse BAC was firstmodified by replacing 714 bp of mouse Cd3d sequence (corresponding topartial sequence of mouse coding exons 2-3 of Cd3d gene) with 939 bp ofhuman CD3D sequence (corresponding to partial sequence of human codingexons 2-3 of CD3D gene) in a single targeting event using a targetingvector comprising a Spec cassette using mouse homology arms.

Mouse BAC comprising a replacement of partial sequence of mouse codingexons 2-3 of CD3d gene with corresponding human sequence wassubsequently modified by replacement of 1,738 bp of mouse Cd3g sequence(corresponding to partial sequence of mouse coding exons 2-4 of Cd3ggene) with 1,639 bp of human CD3G sequence (corresponding to partialsequence of human coding exons 2-4 of CD3G gene) also in a singletargeting event using another Spec cassette-containing vector and mousehomology arms.

Finally, the BAC comprising the replacement of mouse CD3d and CD3g geneswith corresponding human genes was further modified by replacing 6,213bp mouse CD3e sequence with 6,817 bp of human sequence (corresponding toreplacement of partial sequence of mouse coding exons 2 to 4 of mouseCD3e gene with partial sequence of human coding exons 2 to 5 of humanCD3E gene). A 4,996 bp floxed neomycin cassette was inserted upstream ofhuman CD3E sequence knock-in.

The resulting humanized large targeting vector for insertion into EScells is depicted in FIG. 2A, with A, B, C, D, E, F, and G indicatingvarious mouse/human or mouse/NEO cassette or human/NEO cassettejunctions. The sequences at the junctions are depicted in Table 1 below.

TABLE 1 Junctional Sequences of the Large Targeting Vector Sequencedesigna- tion SEQ in FIG. ID 1 Junction Sequence NO: A 5′ mouseCGACTTTCTTGACTTCTATTTGTTA 1 Cd3e/ AACACTGTGCATTCACATCGAATGC XhoI/TAGAAGTTTCCTCGTCCCGCTTCCT (loxP) CCTGAATTGCCTGGGATCCTCTGCT cassetteTGATGCCCTGTAGGAAACGTCCTTT CCTGTGGTATAGAAATGACTG/CTCGAG/ATAACTTCGTATAATGTATGC TATACGAAGTTATATGCATGGCCTCCGCGCCGGGTTTTGGCGCCTCCCGC B 3′ TGTATCTTATCATGTCTGGAATAAC 2 cassetteTTCGTATAATGTATGCTATACGAAG (loxP) TTATGCTAGTAACTATAACGGTCCT IceUI//AAGGTAGCGAGCTAGC//CTTCCAC human AGACACCAATGTTCAAAATGGAGGC CD3ETTGGGGGCAAAATTCTTTTGCTATG TCTCTAGTCGTCCAAAAAATGGTCCTAACTTTTTCTGACTCCTGCTTGTC AAAAATTGTGGGCTCATAGTTAATGC C 3′ humanAGGGGAGAATGGCCTTCATGCACTCC 3 CD3E/ CTCCTCACCTCCAGCGCCTTGTGTTT mouseTCCTTGCTTAGTGATTTCCCCTCTCC Cd3e CCACCCCACCCCCCACAGTGTGTGAGAACTGCATGGAGATGGATGTGATGTC GGTG/GCCATAATCATCATTGTTGACATCTGTATCACTCTGGGCTTGCTGAT GGTCATTTATTACTGGAGCAAGAATAGGAAGGCCAAGGCCAAGCCT D 3′ mouse GAAAGAGAGAGTCTTTCTGCTAACTA 4 Cd3d/ACCCCCAGAAGGCCTTCCGGTCTCAT human GTCCTGCAAAGCAGTAGACGCCCAAA CD3DGCCAGGAGCAGAGTTGCGATGAGGTC AATGAAGATGACACC/AGCCACGGTGGCTGGATCCAGCTCCACACAGCTCTG GCACACTGTGGGGGAAGGGAGGAGAGAGGAGAGGTTGAGAGCCTTTAAGATC AGGGAACCATCCT E 5′ humanCAAGAGAGACAGAAGTCACAAGAAAA 5 CD3D/ AGCCTTCAGAAAGTTCCCCACCAACT SgrDI/GCAGGGGTCAAGGGGGACATGAGGAT mouse GCCATTCAAG/CGTCGACG/AGCGTA Cd3dGGCAGCTTATTGCTCTGCATACTTAC AGACCATTTGTGTAGTAAGGGACATGATGCCGAGTGAAAGGGGCAGGAGCAA CCAGAGGGAGATTTCAGGAAGTTCTCCAGGGACTCGAGGTTCGTGA F 5′ mouse GAAGCCCCACCCAGAAAGGTAGGACAA 6 Cd3g/AGATCATAGTCATATTTACTTCATCCA AsisI/ GGAGAGAAACACAGACACAGCCATTGC humanCTTGGCCATCATCTCTCTCCATCTTGA CD3G CCTCACGTGATCATG/GCGATCGC/GAGTGATTTAGTCTACAATCCGGAAAACT AAGTATAGATACTACCATTTTCATGGATTTGGATCTTTCTTCATCTTGGCCTCA AATAACCATG G 3′ humanGCATTATTGCAGACAGGCAGGAGAAAA 7 CD3G/ CGAACCAGGAAAAACAACTTTCGCAAC mmuseCTGAAGGTTTGTCTCTCCTTTTCCCTA Cd3g CAGTGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCT/GGCTTTATC TTCGCTGAGGTCATCAGCATCTTCTTCCTTGCTCTTGGTGTATATCTCATTGCG GGACAGGATGGACAATACCCTGTCTTA A

The targeted BAC DNA was used to electroporate mouse ES cells comprisinga deletion in mouse CD3 locus to create modified ES cells for generatingmice that express humanized CD3ε, CD3δ, and CD3γ on the surface of theirT cells. ES cells containing insertions of human CD3ε, CD3δ, and CD3γsequences were identified by a quantitative TAQMAN™ assay (see, e.g.,Lie and Petropoulos, 1998. Curr. Opin. Biotechnology 9:43-48,incorporated herein by reference). Specific primer sets and probes weredesigned for detecting insertion of human sequences (gain-of-allele,GOA) and deletion of mouse sequences (loss-of-allele, LOA). Table 2identifies the names and locations of each of primers/probe sets used inthe quantitative PCR assays.

TABLE 2 Primers/Probe Pairs Used for Genotyping Sequence Gene Name AssayFwd Primer Probe (BHQ) Rev Primer Mouse 968 mTU LOA CCTCTGCCATGTGCCGTGATGT GTTCTGAG Cd3e TAGGTTTGTG TTGTTCAATGA AAAGGCGT TAC CCAAATCTTAAGTG (SEQ ID NO: 9) (SEQ ID NO: 10) (SEQ ID NO: 11) Mouse 7164 LOACCAGGCGTACT TGGGCTTACCAT GCTACTCTTC Cd3g mTD TGCTGTTCTG CCAGGACGACCACAAACTG (SEQ ID NO: 12) (SEQ ID NO: 13) CTTAG (SEQ ID NO: 14) Human7170 GOA CCAGCAGTAAG TGTAGAAATGG GGGCTGTGTT CD3E hTU TTCCACTGTTCCTGTGACCCAGCA GCAGTATGAC TAG (SEQ ID NO: 16) (SEQ ID NO: 17)(SEQ ID NO: 15) Human 928 hTU GOA ACCGTGCAAGT ACGTGCTTCCTG TCTCACATCCACD3D TCATTATCGAAG AACCCTTTGGGT GAAGCCCTATC (SEQ ID NO: 18)(SEQ ID NO: 19) (SEQ ID NO: 20) Human 7164 GOA CGAGGGATGTA CACAGAACAAGTGCTCACCAGAA CD3G hTD TCAGTGTAAAG CAAAACCACTCC CAGCAAATACTG GA AAGTG(SEQ ID NO: 23) (SEQ ID NO: 21) (SEQ ID NO: 22)

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0generation mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses NatureBiotech. 25(1):91-99). VELOCIMICE® (F0 mice fully derived from the donorES cell) independently bearing a humanized CD3 genes were identified bygenotyping using a modification of allele assay (see above) that detectsthe presence of the unique human CD3 gene sequences.

The selection cassette may be removed by methods known by the skilledartisan. For example, ES cells bearing the humanized CD3 locus may betransfected with a construct that expresses Cre in order to removefloxed cassette. The selection cassette may optionally be removed bybreeding to mice that express Cre recombinase. Optionally, the selectioncassette is retained in the mice. The mouse/human junction of thehumanized CD3ε allele after selection cassette removal (depicted as A-Bin FIG. 2B), is presented in Table 3 below. The remaining junctionsequences are the same as in the targeting vector and are presented inTable 1 above.

TABLE 3 Junctional Sequences of the Humanized Allele Sequence SEQdesignation ID in FIG. 1B Junction Sequence NO: A-B 5′mouseCGACTTTCTTGACTTCTATTTGTTAAA 8 Cd3e/ CACTGTGCATTCACATCGAATGCTAGA XhoI/AGTTTCCTCGTCCCGCTTCCTCCTGAA Lox/ TTGCCTGGGATCCTCTGCTTGATGCCC IceUI//TGTAGGAAACGTCCTTTCCTGTGGTAT human AGAAATGACTG/CTCGAG/ATAACTTC CD3EGTATAATGTATGCTATACGAAGTTAT/ GCTAGTAACTATAACGGTCCTAAGGTAGCGAGCTAGC//CTTCCACAGACACCA ATGTTCAAAATGGAGGCTTGGGGGCAAAATTCTITTGCTATGICTCTAGTCGTC CAAAAAATGGTCCTAACTITTTCTGACTCCTGCTTGTCAAAAATTGTGGGCTCA TAGTTAATGC

The sequence of the resulting humanized CD3ε, CD3δ, and CD3γ proteins isdepicted in FIG. 3 and included in the sequence listing. Additionally,alignment of mouse-human sequences and junctions at the 5′ and 3′ ofinserted human sequence are shown in FIG. 4 as * and **, respectively.GenBank Protein Accession Numbers for CD3ε, CD3δ, and CD3γ proteins aresummarized below in Table 4.

TABLE 4 GenBank Protein Accession Numbers Mouse Accession # HumanAccession # Protein Name (SEQ ID NO) (SEQ ID NO) CD3ε NP_031674NP_000724 (SEQ ID NO: 27) (SEQ ID NO: 28) CD3δ NP_038515 NP_000723(isoform A) (SEQ ID NO: 29) (SEQ ID NO: 30) CD3γ NP_033980 NP_000064(SEQ ID NO: 31) (SEQ ID NO: 32)

Example 2. Characterization of Humanized CD3 Mice Example 2.1. ImmuneCell Development in Humanized CD3 Mice

Immune cell development in the thymus and periphery of human CD3εδγ micewas assessed using fluorescence-activated cell sorting (FACS) analysisand differential cell counting. Thymus, spleen and lymph nodes wereharvested from cohorts of wildtype (WT, no human CD3γδε), heterozygous(Het, one hCD3γδε allele) and homozygous (Ho, two hCD3γδε alleles) mice.Peripheral blood was obtained by cardiac puncture or retro-orbital bleedinto EDTA coated Microtainer tubes (BD). Single cell suspensions wereprepared from the spleen, LN and thymus using mechanical disruption, andred blood cells were removed from the spleen, thymus and whole blood bylysis with AKC Lysis buffer. Cells were incubated for 10 minutes at roomtemperature with purified antibodies to CD16/CD32 (FcBlock) to blocknon-specific binding via Fc receptors, and then incubated for 30 minutesat 4° C. with a cocktail of directly conjugated antibodies to T and Bcell markers. Cells were washed twice with cold PBS containing 1% BSA,resuspended in buffer and analyzed by flow cytometry on a FACSCanto II™flow cytometer (BD Biosciences). Thymocytes were identified first byforward and side scatter gating, and then by gating on the B220−population. In the periphery, T cells were identified asCD45+/TCRb+/B220−, and B cells were identified as CD45+/TCRb−/B220+.Absolute counts were obtained on a Hemavet 950FS Hematology Analyzer.

As demonstrated in FIG. 5, humanized CD3γδε mice appeared to have normalthymocyte development and normal T cell and B cell ratios in thymus,peripheral blood, and spleen. Additionally, T and B cell percentagesappeared normal in lymph nodes, and absolute cell counts for spleen andlymph nodes (data not shown) were within normal range. CD4 and CD8 cellnumbers in the blood were similar between the WT, Het, and Ho mice.Circulating white blood cells, lymphocytes, monocytes, neutrophils,eosinophils, and basophils all appeared within normal range (data notshown). Thus, normal immune cell development is observed in thehumanized CD3εδγ mice.

Example 2.2. T Cell Response to Infection in Humanized CD3 Mice

To determine whether the humanized CD3 mice (humanized CD3γδε mice)exhibited normal response to infection, the ability of humanized mice toclear lymphocytic choriomeningitis virus (LCMV) was tested. LCMV is amouse tropic virus, where the fate of infection depends on the viralstrain. Infection with Armstrong strain results in an acute infection,where mice can quickly mount a T cell response against the virus andclear the infection in about a week. On the other hand, Clone 13 viruscannot be cleared, and T cells become “exhausted” and chronic infectionis established. As both chronic and acute infections depend on T cellactivity, LCMV is an ideal model to test for T cell function.

6-8 week old humanized CD3 or strain matched control mice were infectedwith 2×10⁵ ffu of Armstrong i.p. and/or 2×10⁶ ffu of Clone 13 i.v. forClone 13 infection, two weeks after infection spleens were harvested andvirus titers were measured by plaque assay. Viral titers were similar inboth control and huCD3 mice (FIG. 6A), indicating that CD3 humanizationdid not have an effect on the T-cell exhaustion phenotype, as T-cellscan control the virus to similar levels in both strains of mice. For theArmstrong strain infection, two weeks after initial Amstrong straininfection, mice were challenged with Clone 13 and two weeks after Clone13 challenge viral titers were measured in spleens. No virus wasdetected in either control or humanized CD3 mice (FIG. 6B). The datasuggests that the acute Armstrong infection was cleared. In addition,this demonstrates that T-cell memory that was elicited from theArmstrong infection was sufficient to protect mice from the subsequentClone 13 infection in both strains of mice.

Example 3. Humanized CD3 Mice as a Model for Testing Anti-CD3-BasedTherapeutic Candidates Example 3.1. Humanized CD3 Mouse for TestingCynomolgus Monkey Cross-Reactive Anti-Human CD3 Antibodies

The ability of different human restricted or cynomolgus cross-reactiveanti-CD3 antibodies to bind splenocytes from wild type (WT) or humanizedCD3γδε (Ho=homozygous, Het=heterozygous) mice was tested usingfluorescence-activated cell sorting (FACS) analysis.

Freshly isolated splenocytes (2×10⁵ per well) were incubated withanti-CD3 antibodies (15 ug/ml) for 30 minutes at 4° C. Post incubation,cells were washed twice and appropriate secondary antibodies (e.g.fluorescent-tagged PE anti-human IgG and directly conjugated antibodiesto T cell markers) were added and incubated for an additional 30 minutesat 4° C., then washed twice. The following antibodies were used:ah/mfCD3-2 and ah/mfCD3-1 are two antibodies that recognize both humanand monkey CD3; ahCD3-2 and ahCD3-1 are two antibodies that onlyrecognize human CD3, amCD3-2C11 is an antibody that recognizes mouse CD3only, control human IgG is an unrelated control antibody, and 2^(nd)only is a secondary antibody only control. Cells were washed twice withcold PBS containing 1% BSA, resuspended in buffer and analyzed by flowcytometry on a FACSCanto II™ flow cytometer (BD Biosciences). T cellswere identified as CD45+/TCRb+/B220−. Anti-mCD3-2C11 engineered tocontain hIgG1 was used to identify T cells on WT mouse splenocytes.

As demonstrated in FIG. 7, anti-CD3 antibodies that recognized onlyhuman CD3 were able to bind CD3 on the surface of splenocytes fromhumanized CD3γδε mice; similarly anti-CD3 antibodies that recognizedhuman and monkey CD3 were able to bind CD3 on the surface of humanizedCD3γδε mice. Thus, mice humanized for all three CD3ε, CD3δ, and CD3γ arerelevant for early pre-clinical studies of CD3-based drug candidateswhich can be followed up by efficacy and toxicity studies in cynomolgusmonkeys.

Example 3.2. T Cell Activation in Humanized CD3 Mice

The ability of anti-human CD3 antibodies to elicit immune response inhumanized CD3 mice was tested. Mice humanized for CD3γδε (n of 2/group),were injected intraperitoneally with 10 ug of different human restrictedor cynomolgus cross-reactive anti-CD3 antibodies (all hIgG1). To obtaincellular composition and plasma cytokine levels, blood was drawn intoEDTA coated Microtainer tubes (BD) from the retro-orbital sinus starting2 hours post injection. The number of peripheral T and B cells wasassessed by FACS. Briefly, 50 ul whole blood was incubated for 30minutes at 4° C. with a cocktail of directly conjugated antibodies to Tand B cell markers. Red blood cells were removed by lysis with AKC Lysisbuffer, and the labeled cells were washed one time with cold PBScontaining 1% BSA. After washing, the cells were re-suspended in coldbuffer and analyzed by flow cytometry on a FACSCANTO II™ flow cytometer(BD Biosciences). T cells were identified as live CD45-F/TCRb+/B220−,and B cells were identified as live CD45+/TCRb−/B220+. Absolute cellcounts were determined by adding a known quantity of CountBright TMAbsolute Counting Beads. Plasma cytokine levels were assessed using aMouse ProInflammatory 7-Plex Ultra-Sensitive Kit (Meso-Scale Discovery)from blood obtained 2 hours post injection.

As demonstrated in FIG. 8A, injection of 10 ug of anti-CD3 antibodiesinduced a transient T and B cell depletion, which was largely restoredby day 4 after initial antibody treatment. Additionally, injection ofanti-CD3 antibodies (both anti-CD3 antibodies recognizing only human CD3(ahCD3-1 and ahCD3-3) and anti-CD3 antibodies recognizing both human andmonkey CD3 (ah/mfCD3-1 and ah/mfCD3-2)) induced cytokine production inCD3γδε humanized mice (FIG. 8B).

In addition, the ability of anti-human CD3, anti-human/cynomolgus CD3,or anti-mouse antibodies to induce proliferation of splenocytes obtainedfrom wild type or humanized CD3γδε mice was assessed using ATP catalyzedquantification (CellTiter Glo®). The activation of mouse splenocytesresults in the release of cytokines, which drive cellular proliferation.Proliferation data was acquired using the following protocol:splenocytes (5×10⁵/well) derived from wild type (WT) or humanizedhomozygous CD3γδε (hCD3γδεHo) were added to 96 well plates which hadbeen coated overnight at 4° C. with decreasing amounts of humanrestricted, cynomolgus cross-reactive, or murine specific anti-CD3antibodies. 500 ng/ml anti-mouse CD28 was added to the cultures, and theplates were incubated for 72 h at 37° C. Following incubation, CellTiterGlo® was added and luminescence was measured using a VICTOR X5multi-label plate reader (PerkinElmer). The EC50 of cell viability (ATPcatalyzed quantification) was determined using Prism (GraphPad Software,San Diego, Calif.). Values were calculated using a 4-parameternon-linear regression analysis.

As demonstrated in FIG. 9, splenocytes from humanized CD3γδε mice wereinduced to proliferate by cynomolgus monkey-crossing CD3 antibodies.

A summary of various properties of WT and CD3γδε mice are presented inFIG. 10. As can be seen, CD3γδε mice are able to bind human CD3 andrespond to anti-human CD3 antibodies, particularly those that are knownto cross-react with monkey CD3, which is an important aspect fortherapeutic agents as preclinical studies on drug candidates are oftenconducted in large animals such as cynomolgus monkeys.

Example 3.3. Tumor Depletion Studies in Humanized CD3 Mouse

Mice humanized for CD3 (humanized CD3γδε mice described above) werehumanized at the CD20 locus such that the mice expressed both humanizedproteins. Humanized CD3/CD20 mice were implanted subcutaneously with2×10⁵ B16F10.9 melanoma tumor cells transduced with human CD20. Startingat Day 0 (day of tumor transplantation), mice were treatedintraperitoneally 2 times per week with either vehicle (PBS; n=5), 0.4mg/kg control Ab 2 (control antibody that does not displaycross-reactivity to CD20 antigen; n=5), 0.4 mg/kg of Ab 1 (anti-CD3/CD20bispecific antibody, see WO2014121087A1, published Aug. 7, 2014, N=5),or 0.004 mg/kg Ab 1 (n=5). Tumor volumes were measured as indicated inFIG. 11A. Mice were sacrificed when tumors reached volume of greaterthan 1500 mm³. As demonstrated in FIG. 11A, treatment with Ab 1 delayedtumor growth when treatment was initiated simultaneously with tumortransplantation.

In a separate experiment, ability of Ab 1 to inhibit tumor growth in analready established tumor was also tested (FIG. 11B). Humanized CD3/CD20mice were implanted subcutaneously with 2×10⁵ B16F10.9 melanoma tumorcells expressing human CD20. On day 10 post tumor implantation, micewere randomized based on tumor size and organized into the followingtreatment groups, 5 mice in each group: vehicle (PBS), 4 mg/kg controlAb 2 (control antibody that does not display cross-reactivity to CD20antigen), 4 mg/kg of Ab 1, or 0.04 mg/kg Ab 1. All mice were treatedi.p. 2 times a week. Mice were sacrificed when tumors reached volume ofgreater than 1500 mm³. As demonstrated in FIG. 11B, treatment with Ab 1delayed tumor growth of already established tumors, demonstrating thatthe humanized CD3 mice are advantageous for early drug candidatestudies.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Entire contents of all non-patent documents, patent applications andpatents cited throughout this application are incorporated by referenceherein in their entireties.

1. A genetically modified non-human animal comprising an endogenousnon-human CD3 locus genetically modified to encode an extracellulardomain of a human CD3 protein, wherein the human CD3 protein is CD3ε,CD3δ, CD3γ, CD3ζ, or any combination thereof.
 2. The animal of claim 1,wherein the endogenous non-human CD3 locus is genetically modified toencode an extracellular domain of human CD3ε, an extracellular domain ofhuman CD3δ, and an extracellular domain of human CD3γ.
 3. The animal ofclaim 1, wherein the endogenous non-human CD3 locus is geneticallymodified not to express functional extracellular domain(s) of non-humanCD3 protein(s) corresponding to the human CD3 protein(s).
 4. The animalof claim 1, wherein the endogenous locus further encodes transmembraneand cytoplasmic domains of a CD3 protein of the endogenous non-humananimal, wherein the animal expresses a chimeric CD3 protein on thesurface of its T-cells comprising the extracellular domain of the humanCD3 protein and the transmembrane and cytoplasmic domains of theendogenous non-human animal CD3 protein.
 5. The animal of claim 2,wherein the animal comprises: at an endogenous CD3ε locus a nucleic acidsequence encoding an extracellular domain of a human CD3ε operablylinked to a nucleic acid sequence encoding transmembrane and cytoplasmicdomains of CD3ε of the endogenous non-human animal, at an endogenousCD3δ locus a nucleic acid sequence encoding an extracellular domain of ahuman CD3δ operably linked to a nucleic acid sequence encodingtransmembrane and cytoplasmic domains of CD3δ protein of the endogenousnon-human animal, and at an endogenous CD3γ locus a nucleic acidsequence encoding an extracellular domain of a human CD3γ operablylinked to a nucleic acid sequence encoding transmembrane and cytoplasmicdomains of an CD3γ protein of an endogenous non-human animal CD3γ;wherein the non-human animal expresses chimeric CD3ε, CD3δ, and CD3γproteins on the surface of its T cells.
 6. The animal of claim 1 whereinthe animal comprises extracellular domains of human CD3 proteins whichcomprise the sequences of SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35.7. The animal of claim 1, wherein the animal is a mammal.
 8. The animalof claim 1, wherein the animal is a rodent.
 9. The animal of claim 8,wherein the animal is a mouse.
 10. The mouse of claim 9, wherein themouse comprises: at an endogenous mouse CD3ε locus a nucleic acidsequence encoding an extracellular domain of a human CD3ε operablylinked to a nucleic acid sequence encoding transmembrane and cytoplasmicdomains of an endogenous mouse CD3ε, at an endogenous mouse CD3δ locus anucleic acid sequence encoding an extracellular domain of a human CD3δoperably linked to a nucleic acid sequence encoding transmembrane andcytoplasmic domains of an endogenous mouse CD3δ, and at an endogenousmouse CD3γ locus a nucleic acid sequence encoding an extracellulardomain of a human CD3γ operably linked to a nucleic acid sequenceencoding transmembrane and cytoplasmic domains of an endogenous mouseCD3γ, and wherein the mouse expresses chimeric CD3ε, CD3δ, and CD3γproteins on the surface of its T cells.
 11. The mouse of claim 10,wherein the amino acid sequence of the chimeric CD3ε protein is setforth in SEQ ID NO:24, the amino acid sequence of the chimeric CD3δprotein is set forth in SEQ ID NO:25, and the amino acid sequence of thechimeric CD3γ protein is set forth in SEQ ID NO:26.
 12. The animal ofclaim 1, which is heterozygous for the modified endogenous non-human CD3locus.
 13. The animal of claim 1, which is homozygous for the modifiedendogenous non-human CD3 locus.
 14. A method of making a geneticallymodified non-human animal as defined in claim 1, comprising: introducinga nucleic acid sequence encoding an extracellular domain of human CD3protein, wherein the human CD3 protein is CD3ε, CD3δ, CD3γ, CD3ζ, or anycombination thereof into the genome of a cell of non-human animal at anendogenous CD3 locus; and propagating the genetically modified non-humananimal from the cell.
 15. The method of claim 14, wherein the cell is asingle ES cell, and the single ES cell is introduced into a mouse embryoto propagate a mouse.
 16. A mouse model for testing a CD3-basedbispecific antigen-binding protein, wherein the antigen-binding proteinis capable of binding both CD3 and a non-mouse antigen of interest,comprising: a genetically modified mouse as defined by claim 9 furthercomprising a cell expressing or comprising the non-mouse antigen ofinterest.
 17. (canceled)
 18. A method of screening drug candidates thattarget an antigen of interest comprising: a. introducing into agenetically modified mouse as defined by claim 9 the antigen ofinterest, b. contacting said mouse with a drug candidate of interest,wherein the drug candidate is directed against the human CD3 and theantigen of interest, and c. determining if the drug candidate isefficacious in preventing, reducing or eliminating cells characterizedby the presence or expression of the antigen of interest.
 19. The methodof claim 18, wherein the step of introducing comprises expressing in themouse the antigen of interest.
 20. The method of claim 18, wherein thestep of introducing comprises infecting the mouse with the antigen ofinterest.
 21. The method of claim 19, wherein the step of expressing inthe mouse the antigen of interest comprises genetically modifying themouse to express the antigen of interest.
 22. The method of claim 18,wherein the step of introducing comprises introducing into said mouse acell expressing the antigen of interest.
 23. The method of claim 16,wherein the cell is a tumor cell.
 24. The method of claim 22, whereinthe cell is a bacterial cell.
 25. The method of claim 20, wherein theinfecting comprising performing viral or bacterial infection.
 26. Themethod of claim 18, wherein the mouse is an immunocompetent mouse. 27.The method of claim 18, wherein the antigen of interest is a tumorassociated antigen.
 28. The method of claim 27, wherein the tumorassociated antigen is selected from the group consisting of ALK, BAGEproteins, BIRC5 (survivin), BIRC7, CA9, CALR, CCR5, CD19, CD20 (MS4A1),CD22, CD27, CD30, CD33, CD38, CD40, CD44, CD52, CD56, CD79, CDK4,CEACAM3, CEACAM5, CLEC12A, EGFR, EGFR variant III, ERBB2 (HER2), ERBB3,ERBB4, EPCAM, EPHA2, EPHA3, FCRL5, FLT3, FOLR1, GAGE proteins, GD2, GD3,GPNMB, GM3, GPR112, IL3RA, KIT, KRAS, LGR5, EBV-derived LMP2, L1CAM,MAGE proteins, MLANA, MSLN, MUC1, MUC2, MUC3, MUC4, MUC5, MUC16, MUM1,ANKRD30A, NY-ESO1 (CTAG1B), OX40, PAP, PAX3, PAX5, PLAC1, PRLR, PMEL,PRAME, PSMA (FOLH1), RAGE proteins, RET, RGS5, ROR1, SART1, SART3,SLAMF7, SLC39A6 (LIV1), STEAP1, STEAP2, TERT, TMPRSS2, Thompson-nouvelleantigen, TNFRSF17, TYR, UPK3A, VTCN1, and WT1. 29-30. (canceled)
 31. Themethod of claim 18, wherein the antigen is a viral antigen selected fromthe group consisting of HIV; hepatitis A; hepatitis B; hepatitis C;herpes virus such as HSV-1, HSV-2, CMV, HAV-6, VZV, and Epstein Barrvirus; adenovirus; influenza virus; flavivirus; echovirus; rhinovirus;coxsackie virus; coronavirus; respiratory syncytial virus; mumps virus;rotavirus; measles virus; rubella virus; parvovirus; vaccinia virus;HTLV; dengue virus; papillomavirus; molluscum virus; poliovirus; rabiesvirus; JC virus; ebola virus; and arboviral encephalitis virus antigen.32. (canceled)
 33. The method of claim 18, wherein the antigen is abacterial antigen selected from the group consisting of chlamydia,rickettsia, mycobacteria, staphylococci, streptococci, pneumonococci,meningococci, gonococci, klebsiella, proteus, serratia, pseudomonas,legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism,anthrax, plague, leptospira, and Lyme disease bacterial antigen.
 34. Themethod of claim 18, wherein the drug candidate is an antibody or anantigen-binding protein. 35-36. (canceled)
 37. The method of claim 34,wherein the antibody or the antigen-binding protein is a bispecificantibody or a bispecific antigen-binding protein, respectively, which iscapable of binding both human CD3 protein and the antigen of interest.38. The method of claim 18, wherein the drug candidate is capable ofrecognizing a monkey CD3 protein. 39-44. (canceled)